Preparation of a therapeutic composition for treating autoimmune diseases

ABSTRACT

The present invention provides a composition comprising dendritic cells loaded with hHsp60sp, which dendritic cells are from a subject and have been fixed with paraformaldehyde (PFA). The subject may suffer from an autoimmune disease. Also provided are a method for preparing the composition; recombinant human cells comprising a heterologous gene encoding a fusion protein of HLA-E and hHsp60sp or B7sp, and expressing the fusion protein on the surface of the cells; a method for determining a percentage of maximum inhibition of testing the function of the HLA-E restricted CD8+ Treg cells from a subject, determining whether HLA-E restricted CD8+ Treg cells freshly isolated from a subject are defective, or determining whether defective HLA-E restricted CD8+ Treg cells from a subject are correctable; and a method for correcting defective HLA-E restricted CD8+ Treg cells, treating type 1 diabetes (T1D), or treating multiple sclerosis (MS).

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.63/195,087, filed May 31, 2021, and U.S. Provisional Application No.63/297,354, filed Jan. 7, 2022, the contents of each of which areincorporated herein by reference in their entireties for all purposes.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledAVO-102US_SequenceListing.txt, created May 26, 2022, which is 1,233bytes in size. The information in the electronic format of the SequenceListing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to dendritic cells derived from a subject, whichwere loaded with hHsp60sp and fixed with paraformaldehyde, and usesthereof for correcting defective regulatory CD8+ T cells in the samesubject and/or treating one or more autoimmune diseases in the samesubject.

BACKGROUND OF THE INVENTION

Autoimmune diseases result from breakdown in mechanisms that maintainunresponsiveness to self. For example, type 1 diabetes (T1D) is anautoimmune disorder in which T cells are self-reactive or activated Tcells that are activated against major antigenic components ofpancreatic β cells are out of the control by the normal regulatorymechanisms. While these self-reactive T cells are under the control ofperipheral regulatory mechanisms in healthy individuals, failure ofcontrol leads to destruction of the β cells and subsequent T1D. Theautoimmune attack on pancreatic β cells is orchestrated by a variety ofcells that either by directly killing the β cells or, indirectly, byproducing cytokines and other toxic mediators to damage the β cells. Thefacts that these self-reactive T cells work together with otherlymphocytes and antigen-presenting cells to mediate this damage havebeen shown in animal models to be important both in the early stages ofdiabetes development and in the final effector stages.

Selective control of unwanted immune responses could be achieved byinduction of “antigen-specific tolerance”, provided that the specificantigens that elicit the unwanted immune responses have been identified.However, induction of “antigen specific tolerance” requires preciseknowledge of the peptide associated with particular MHC molecules andthe highly polymorphic human HLA system inevitably increase theuncertainty and puts a huge burden on searching for such a MHC/peptidecomplex in each patient. Thus, the necessity of identification of aprecise MHC/peptide complex for each individual from an unknown andcountless antigen pool makes the induction of “antigen-specifictolerance” an unfeasible approach or may be an “impossible mission” tospecifically and effectively treat organ specific autoimmune diseasesand/or control graft rejection in clinical immunology.

HLA-E restricted CD8+ regulatory T cells, provide a simple and unified“class action” of self-nonself discrimination, regulating the unwantedanti-self immune responses, independent of the knowledge of anyparticular pathogenic antigens, which are largely undetermined, inautoimmune diseases and/or graft rejection.

A subset of HLA-E restricted regulatory CD8+ T cells are known toselectively down-regulate all potentially pathogenic self-reactive Tcells by specifically recognizing a common target structure, i.e., afamily of peptides, represented by Hsp60sp, associated with HLA-Eexpressed on pathogenic self-reactive T cells. Such pathogenicself-reactive T cells are responsible for a variety of autoimmunediseases. The dysfunction or failure of this Q/E CD8+ Treg cell subsetto recognize, or to suppress, its target population results in apermissive state, in which organ-specific autoimmunity may emerge.

The HLA-E restricted regulatory CD8+ T cells selectively down-regulatethe self-reactive T cell pool by specifically recognizing a Biomarker(HLA-E/Hsp60sp peptide complex) preferentially expressed on theself-reactive T cells regardless of which antigens activated the targetT cells. Such unique feature presents a rarely seen therapeuticadvantage to target a wide spectrum of autoimmune diseases and otherimmunologically relevant conditions without the necessity to identifythe disease specific pathogenic antigens, which is an extremelychallenging task currently.

U.S. Pat. No. 9,421,249 (“the '249 patent”) discloses a murinecomposition for activation of Qa-1restricted CD8+ T cells so as tosuppress murine autoreactive T cells. The composition of the '249 patentcomprises a murine dendritic cell loaded extracellularly with a murineHsp60sp peptide. The association of dendritic cells with human Hsp60sppeptide is known unstable. Further, Jiang (JCI 120(10):3641-3650 (2010))discloses that a majority of people with Type 1 diabetes who were testedin the study were found to have a defect in CD8+ T cell recognition ofHLA-E/hHsp60sp, which was associated with failure of self/non-selfdiscrimination, and the defect in the CD8+ T cells from most of the T1Dpatients tested could be corrected in vitro by exposure to autologousimmature dendritic cells loaded with hHsp60sp in vitro. However, noclinical use of autologous immature dendritic cells loaded with hHsp60sphas been performed to correct the defect in HLA-E restricted CD8+ Tcells when administered to a human individual (e.g., patient). It wasunknown whether other autoimmune patients have defective HLA-Erestricted CD8+ T cells that are correctable in vitro by autologousimmature dendritic cells loaded with hHsp60sp. Nor has there been anyclinical use of any autologous immature dendritic cells loaded withhHsp60sp, let alone with demonstrated stability and efficacy forcorrecting defective HLA-E restricted CD8+ Treg cells or treating anautoimmune disease (e.g., T1D) in a human individual (e.g., patient).

Table 1 summarizes the dose regimens and routes of administration ofdendritic cell-based therapies in some human clinical trials, which havebeen determined to be safe for use.

TABLE 1 Dendritic cell-based therapies Therapy to Dose/schedule RouteNumber Subjects NHL 2-17 × 10⁶/4 weeks × 4 i.v. 35 (Non-Hodgkin'slymphoma) 2-17 × 10⁶/4 weeks × 4 i.v. 10 Melanoma 5-50 × 10⁶/2 weeks × 4i.v./i.d./i.n. 28 5-50 × 10⁶/2 weeks × 4 i.v. 14 3 × 10⁶ + 6/12 × 10⁶/2weeks × 5 i.d. + i.v. 13 Myeloma 0.5-11 × 10⁶/4 weeks × 2 i.v. 12 3.5-89× 10⁶/2 weeks × 3 i.v. 6 Prostate cancer 1-20 × 10⁶/6-8 weeks × 4-5 i.v.51 10 × 10⁶/2 weeks × 4 i.v. + i.d. 8 0.3-40 × 10⁶/4 weeks × 2i.v./i.d./i.l. 21 10 × 10⁶/2 weeks × 3 i.v. + i.d. 13 0.94-2.02 × 10⁸/ 4weeks × 3 i.v. 14 RCC 5-10 × 10⁶/4 weeks × 3-13 i.v./i.d. 35 (Renal cellcarcinoma) 10-50 × 10⁶ + 10 × 106/2weeks × 3 i.v. + i.d. 10 Type 1diabetes 10 × 10⁶/2 weeks × 4 i.d. 10

There remains a need for stable autologous dendritic cells loaded withhHsp60sp capable of correcting defective HLA-E restricted CD8+ T cellsor treating an autoimmune disease such as, e.g. T1D, in a subject inneed thereof and reliable methods for identifying a subject havingdefective HLA-E restricted CD8+ Treg cells that are correctable by theautologous dendritic cells loaded with hHsp60sp.

SUMMARY OF THE INVENTION

The present invention provides dendritic cells (DCs) prepared from asubject and loaded with human heat shock protein 60 signal peptide(hHsp60sp), also known as hHsp60sp pulsed DCs and uses thereof. Theinventor has discovered that fixation of the hHsp60sp pulsed DCs with 2%paraformaldehyde (PFA) stabilizes the association between the DCs andthe hHsp60sp. The resulting fixed hHsp60sp pulsed DCs are referred to aspDC(H)s. The inventor has also surprisingly discovered that theautologous pDC(H)s are therapeutically effective for treating a subjectsuffering from an autoimmune disease such as, for example, type 1diabetes (T1D), multiple sclerosis (MS), psoriasis, rheumatoidarthritis, lupus, vitiligo, pemphigus or dermatomyositis, and improvingbiological indicators associated with T1D in the T1D patients up toone-two year/s after treatment. The inventor has further generated novelhuman HLA-A/B/C—deficient B cell lines having stable surface expressionof a complex of HLA-E and the hHsp60sp (TH1) or a complex of HLA-E andB7sp (TB1), enabling a reliable potency assay for identifying a subjecthaving defective HLA-E restricted CD8+ Treg cells that are correctableby the treatment of autologous pDC(H)s.

A composition comprising dendritic cells loaded with hHsp60sp isprovided. The dendritic cells are from a subject in need of thedendritic cells loaded with hHsp60sp, and the dendritic cells loadedwith hHsp60sp have been fixed with paraformaldehyde (PFA).

In the composition, the dendritic cells loaded with hHsp60sp may be in atherapeutically effective amount for correcting correctable defect ofHLA-E restricted CD8+ Treg cells from a subject. The composition may beformulated for intravenous administration to the subject.

In the composition, the dendritic cells loaded with hHsp60sp may be in atherapeutically effective amount for treating an autoimmune disease in asubject. The composition may be formulated for intravenousadministration to the subject.

The composition may further comprise a medium. The medium may comprisedimethyl sulfoxide (DMSO), human serum albumin (HAS) and plasmalyte-A.

The composition may further comprise greater than 80% total CD11c+(gatedon large cells).

The composition may further comprise a cryoprotectant.

According to the composition of the present invention, the subject maysuffer from an autoimmune disease. The autoimmune disease may beselected from the group consisting of type 1 diabetes (T1D), multiplesclerosis (MS), psoriasis, rheumatoid arthritis, lupus, vitiligo,pemphigus and dermatomyositis. The autoimmune disease may be type 1diabetes (T1D). The autoimmune disease may be multiple sclerosis (MS).

For each composition of the present invention, a method is provided forpreparing the composition. The preparation method comprises (a)isolating mononuclear cells from a subject; (b) incubating themononuclear cells in a culture for no more than six days to produceimmature dendritic cells (DCs); (c) harvesting the immature DCs from theculture in step (b); (d) incubating the harvested DCs with hHsp60sp toproduce hHsp60sp loaded dendritic cells (DCs); (e) fixing the hHsp60sploaded DCs with paraformaldehyde (PFA) to produce fixed hHsp60sp loadedDCs; and (f) suspending the hHsp60sp loaded DCs in a medium, whereby thecomposition is prepared. The preparation method may further comprisefreezing the composition.

According to the preparation method of the present invention, thehHsp60sp loaded DCs may be fixed with 2% PFA in step (d). Thecomposition may comprise greater than 80% total CD11c+(gated on largecells). The medium may comprise dimethyl sulfoxide (DMSO), human serumalbumin (HAS) and plasmalyte-A. The composition may further comprise apharmaceutically acceptable carrier. The composition may furthercomprise a cryoprotectant. The mononuclear cells may be cultured in thepresence of GM-CSF and IL-4 in step (b).

According to the preparation method of the present invention, thecomposition may be formulated for intravenous administration to thesubject. The subject may suffer from an autoimmune disease. Theautoimmune disease may be selected from the group consisting of type 1diabetes (T1D), multiple sclerosis (MS), psoriasis, rheumatoidarthritis, lupus, vitiligo, pemphigus and dermatomyositis. Theautoimmune disease may be type 1 diabetes (T1D). The autoimmune diseasemay be multiple sclerosis (MS).

For each preparation method of the present invention, a compositionprepared according to the method is provided.

A first recombinant human cell is provided. The first recombinant humancell comprises a heterologous gene encoding a first fusion protein andexpresses the fusion protein on the surface of the first recombinantcell. The fusion protein may comprise human leukocyte antigen system E(HLA-E) and hHsp60sp. The recombinant cell may express the fusionprotein permanently. The fusion protein may further comprise a linkerbetween the HLA-E and the hHsp60sp.

A cell line having an ATCC accession number of PTA-127256 is provided.

A second recombinant human cell is provided. The second recombinanthuman cell comprises a heterologous gene encoding a fusion protein andexpresses the fusion protein on the surface of the second recombinanthuman cell. The fusion protein comprises a human leukocyte antigensystem E (HLA-E) and B7sp. The second recombinant human cell expressesthe fusion protein permanently. The fusion protein further comprising alinker between the HLA-E and the B7sp.

A cell line having an ATCC accession number of PTA-127257 is provided.

A method is provided for determining a percentage of maximum inhibitionof testing the function of the HLA-E restricted CD8+ Treg cells from asubject. The function determination method comprises (a) determining apercentage of maximum inhibition for specific target cells, wherein thespecific target cells are cells expressing a complex of HLA-E andhHsp60sp on the surface of the specific target cells. Step (a) comprises(i) obtaining a specific target cell mixture having an equal number ofthe specific target cells and unloaded target cells, wherein theunloaded target cells are cells expressing HLA-E on the surface of theunloaded target cells; (ii) culturing the specific target cell mixturein the absence of the testing HLA-E CD8+ Treg cells; (iii) quantifyingthe ratio of the specific target cells over the unloaded target cellsafter being cultured in the absence of the testing HLA-E restricted CD8+Treg cells to calculate a first control ratio as the ratio of thequantified proliferation of the specific target cells to the quantifiedproliferation of the unloaded target cells after being cultured in theabsence of the testing HLA-E restricted CD8+ Treg cells; (iv) culturingthe specific target cell mixture in the presence of the testing HLA-Erestricted CD8+ Treg cells at graded ratios of the testing HLA-Erestricted CD8+ Treg cells to the specific target cells (specific E/Tratios); (v) quantifying proliferation of the specific target cells andthe unloaded target cells after being cultured in the presence of thetesting HLA-E restricted CD8+ Treg cells to calculate a firstexperimental ratio as the ratio of the quantified proliferation of thespecific target cells to the quantified proliferation of the unloadedtarget cells after being cultured in the presence of the testing HLA-Erestricted CD8+ Treg cells at each of the graded specific E/T ratios;and (vi) calculating a percentage of specific inhibition for thespecific target cells at each of the graded specific E/T ratios as(first control ratio−first experimental ratio)/first control ratio×100%,wherein the percentage of maximum inhibition for the specific targetcells is the highest value of the percentages of specific inhibition forthe specific target cells at the graded specific E/T ratios. Thefunction determination method further comprises (b) determining apercentage of maximum inhibition for control target cells, wherein thecontrol target cells are cells expressing the HLA-E and B7sp on thesurface of the control target cells. Step (b) comprises (i) obtaining acontrol target cell mixture having an equal number of the unloadedtarget cells and the control target cells; (ii) culturing the controltarget cell mixture in the absence of the testing HLA-E CD8+ Treg cells;(iii) quantifying proliferation of the control target cells and theunloaded target cells after being cultured in the absence of the testingHLA-E restricted CD8+ Treg cells to calculate a second control ratio asthe ratio of the quantified proliferation of the control target cells tothe quantified proliferation of the unloaded target cells after beingcultured in the absence of the testing HLA-E CD8+ Treg cells; (iv)culturing the control target cell mixture in the presence of the testingHLA-E restricted CD8+ Treg cells at graded ratios of the testing HLA-Erestricted CD8+ Treg cells to the control target cells (control E/Tratios); (v) quantifying proliferation of the control target cells andthe unloaded target cells after being cultured in the presence of thetesting HLA-E restricted CD8+ Treg cells to calculate a secondexperimental ratio as the ratio of the quantified proliferation of thecontrol target cells to the quantified proliferation of the unloadedtarget cells after being cultured in the presence of the testing HLA-Erestricted CD8+ Treg cells at each of the graded specific E/T ratios;and (vi) calculating a percentage of specific inhibition for the controltarget cells at each of the graded control E/T ratios as (second controlratio−second experimental ratio)/second control ratio×100%, wherein thepercentage of maximum inhibition for the control target cells is thehighest value of the percentages of specific inhibition for the controltarget cells at the graded control E/T ratios. The functiondetermination method further comprises (c) calculating the percentage ofthe maximum specific inhibition for the testing HLA-E restricted CD8+Treg cells by subtracting the percentage of maximum inhibition for thecontrol target cells from the percentage of maximum inhibition for thespecific target cells.

According to the function determination method of the present invention,the specific target cells may be from the cell line having an ATCCaccession number of PTA-127256 and the control target cells may be fromthe cell line having an ATCC accession number of PTA-127257. The subjectmay suffer from an autoimmune disease. The autoimmune disease may beselected from the group consisting of type 1 diabetes (T1D), multiplesclerosis (MS), psoriasis, rheumatoid arthritis, lupus, vitiligo anddermatomyositis. The subject may suffer from type 1 diabetes (T1D). Thesubject may suffer from multiple sclerosis (MS).

A method is provided for determining whether HLA-E restricted CD8+ Tregcells freshly isolated from a subject are defective. The defectdetermination method comprises determining a percentage of maximuminhibition for the freshly isolated HLA-E restricted CD8+ Treg cellsaccording to the function determination method using the freshlyisolated HLA-E restricted CD8+ Treg cells as the testing HLA-E CD8+ Tregcells. A percentage of maximum inhibition less than 50% of the freshlyisolated HLA-E CD8+ Treg from a normal healthy people indicates that thefreshly isolated HLA-E restricted CD8+ Treg cells are defective and thesubject has defective HLA-E CD8+ Treg cells.

According to the defect determination method of the present invention,the specific target cells may be from the cell line having an ATCCaccession number of PTA-127256 and the control target cells may be fromthe cell line having an ATCC accession number of PTA-127257. The subjectmay suffer from an autoimmune disease. The autoimmune disease may beselected from the group consisting of type 1 diabetes (T1D), multiplesclerosis (MS), psoriasis, rheumatoid arthritis, lupus, vitiligo anddermatomyositis. The subject may suffer from type 1 diabetes (T1D). Thesubject may suffer from multiple sclerosis (MS).

A method is provided for determining whether defective HLA-E restrictedCD8+ Treg cells from a subject are correctable. The correctablenessdetermination method comprises (a) activating the defective HLA-Erestricted CD8+ Treg cells with autologous dendritic cells loaded withhHsp60sp to produce CD8(H) cells; (b) determining a percentage ofmaximum inhibition for the CD8(H) cells according to the functiondetermination method using the CD8(H) cells as the testing HLA-E CD8+Treg cells; (c) activating the defective HLA-E restricted CD8+ Tregcells with fixed autologous dendritic cells loaded with B7sp to produceCD8(B) cells; (d) determining a percentage of maximum inhibition for theCD8(B) cells according to the function determination method using theCD8(B) cells as the testing HLA-E CD8+ Treg cells; and (e) calculating anormalized percentage of maximum inhibition for the defective HLA-Erestricted CD8+ Treg cells by subtracting the percentage of maximuminhibition for the CD8(B) cells from the percentage of maximuminhibition for the CD8(H) cells. A normalized percentage of maximuminhibition greater than 50% of the HLA-E restricted CD8+ Treg cells fromnormal healthy control people indicates that the defective HLA-Erestricted CD8+ Treg cells are correctable and the subject hascorrectable defective HLA-E CD8+ Treg cells.

According to the correctableness determination method of the presentinvention, the specific target cells may be from the cell line having anATCC accession number of PTA-127256 and the control target cells may befrom the cell line having an ATCC accession number of PTA-127257. Thedefective HLA-E restricted CD8+ Treg cells from the subject may bedetermined according to the defect determination method. The subject maysuffer from an autoimmune disease. The autoimmune disease may beselected from the group consisting of type 1 diabetes (T1D), multiplesclerosis (MS), psoriasis, rheumatoid arthritis, lupus, vitiligo anddermatomyositis. The subject may suffer from type 1 diabetes (T1D). Thesubject may suffer from multiple sclerosis (MS).

A method is provided for correcting defective HLA-E restricted CD8+ Tregcells in a subject in need thereof, wherein the defective HLA-Erestricted CD8+ Treg cells are correctable. The correction methodcomprises administering to the subject a therapeutically effectiveamount of a therapeutic composition. The therapeutic compositioncomprises fixed autologous dendritic cells loaded with the hHsp60sp in amedium. The dendritic cells loaded with hHsp60sp have been fixed withparaformaldehyde (PFA).

According to the correction method of the present invention, the mediummay comprise dimethyl sulfoxide (DMSO), human serum albumin (HSA) andplasmalyte-A. The therapeutic composition may further comprise greaterthan 80% total CD11c+(gated on large cells). The therapeutic compositionmay further comprise a cryoprotectant. The therapeutic composition maybe administered to the subject intravenously. The subject may sufferfrom an autoimmune disease. The autoimmune disease may be selected fromthe group consisting of type 1 diabetes (T1D), multiple sclerosis (MS),psoriasis, rheumatoid arthritis, lupus, vitiligo and dermatomyositis.The subject may suffer from type 1 diabetes (T1D). The subject maysuffer from multiple sclerosis (MS).

A method is provided for treating type 1 diabetes (T1D) in a subject inneed thereof. The T1D treatment method comprises administering to thesubject a therapeutically effective amount of a therapeutic composition.The therapeutic composition comprises autologous dendritic cells loadedwith hHsp60sp in a medium. The dendritic cells loaded with hHsp60sp havebeen fixed with paraformaldehyde (PFA).

According to the T1D treatment method of the present invention, themedium may comprise dimethyl sulfoxide (DMSO), human serum albumin (HAS)and plasmalyte-A. The therapeutic composition may further comprisegreater than 80% total CD11c+(gated on large cells). The therapeuticcomposition may further comprise a cryoprotectant. The therapeuticcomposition may be administered to the subject intravenously.

According to the T1D treatment method of the present invention, thesubject may be diagnosed with T1D not more than three months prior tothe treatment. The subject may be diagnosed with T1D not more than 12months prior to the treatment. The subject may be a minor. The subjectmay have correctable defective HLA-E restricted CD8+ Treg cells.

A method is provided for treating multiple sclerosis (MS) in a subjectin need thereof. The MS treatment method comprises administering to thesubject a therapeutically effective amount of a therapeutic composition.The therapeutic composition comprises autologous dendritic cells loadedwith hHsp60sp in a medium. The dendritic cells loaded with hHsp60sp havebeen fixed with paraformaldehyde (PFA).

According to the MS treatment method of the present invention, themedium may comprise dimethyl sulfoxide (DMSO), human serum albumin (HAS)and plasmalyte-A. The therapeutic composition may further comprisegreater than 80% total CD11c+(gated on large cells). The therapeuticcomposition may further comprise a cryoprotectant. The therapeuticcomposition may be administered to the subject intravenously. Thesubject may have correctable defective HLA-E restricted CD8+ Treg cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show results from a CD8+ Treg cell specificity assay as setforth in Example 8. The CD8+ Treg cells from most of clinicallydiagnosed multiple sclerosis (MS) patients, whom were randomly tested,both RRMS (relapsing-remitting multiple sclerosis) and SPMS(secondary-progressive multiple sclerosis), were functionally defectivewhen compared with the normal functional CD8+ Treg cells from healthyindividuals prior to treatment with the therapeutic agent of the presentinvention, whereas the function of the defective CD8+ Treg cells fromthe majority of the MS patients tested was corrected or restored afterthe treatment. A. BEFORE therapy, the readings between “normal” (N=16)and “RRMS/SPMS patients” (N=32) are statistically significant, P<0.0001.B. The “control” group showed normal pathway function BEFORE therapy(N=16), and statistically non-distinguishable AFTER therapy (N=12),P=0.1392. C. The “patient” group (N=32) showed defective pathwayfunction BEFORE therapy, and corrected defect AFTER therapy, showingstatistically significant on the effect of the treatment, P<0.0001.

FIGS. 2A-2C show results from a CD8+ Treg cell specificity assay as setforth in Example 9. The CD8+ Treg cells from psoriasis patients werefunctionally defective when compared with the normal functional CD8+Treg cells from healthy individuals prior to treatment with thetherapeutic agent of the present invention, whereas the function of thedefective CD8+ Treg cells from the psoriasis patients was corrected orrestored after the treatment. A. BEFORE therapy, the readings between“normal” (N=16) and “Psoriasis patients” (N=42) are statisticallysignificant, P<0.0001. B. The “control” group showed normal pathwayfunction BEFORE therapy (N=16), and statistically non-distinguishableAFTER therapy (N=12), P=0.1392. C. The “patient” group (N=42) showeddefective pathway function BEFORE therapy, and the defect was correctedAFTER therapy, showing statistically significant on the effect of thetreatment, P<0.0001.

FIGS. 3A-3C show results from a CD8+ Treg cell specificity assay as setforth in Example 10. The CD8+ Treg cells from rheumatoid arthritis (RA)patients are functionally defective when compared with the normalfunctional CD8+ Treg cells from healthy individuals prior to treatmentwith the therapeutic agent of the present invention, whereas thefunction of the defective CD8+ Treg cells from the RA patients wascorrected or restored after the treatment. A. BEFORE therapy, thereadings between “normal” (N=16) and “RA” patients” (N=15) arestatistically significant, P<0.0001. B. The “control” group showednormal pathway function BEFORE therapy (N=16), and statisticallynon-distinguishable AFTER therapy (N=12), P=0.1392. C. The “patient”group (N=15) showed defective pathway function BEFORE therapy, and thedefect was corrected AFTER therapy, showing statistically significant onthe effect of the treatment, P<0.0001.

FIGS. 4A-4C show results from a CD8+ Treg cell specificity assay as setforth in Example 11. The CD8+ Treg cells from lupus patients arefunctionally defective when compared with the normal functional CD8+Treg cells from healthy individuals prior to treatment with thetherapeutic agent of the present invention, whereas the function of thedefective CD8+ Treg cells from the lupus patients was corrected orrestored after the treatment. A. BEFORE therapy, the readings between“normal” (N=16) and “lupus patients” (*N=31) are statisticallysignificant, P<0.0001. B. The “control” group showed normal pathwayfunction BEFORE therapy (N=16), and statistically non-distinguishableAFTER therapy (N=12), P=0.1392. C. The “patient” group (N=31) showeddefective pathway function BEFORE therapy, and the defect was correctedAFTER therapy, showing statistically significant on the effect of thetreatment, P<0.0001.

FIGS. 5A-5C show results from a CD8+ Treg cell specificity assay as setforth in Example 12. The CD8+ Treg cells from vitiligo patients arefunctionally defective when compared with the normal functional CD8+Treg cells from healthy individuals prior to treatment with thetherapeutic agent of the present invention, whereas the function of thedefective CD8+ Treg cells from the vitiligo patients was corrected orrestored after the treatment. A. BEFORE therapy, the readings between“normal” (N=16) and “patients” (N=27) are statistically significant,P<0.0001. B. The “control” group showed normal pathway function BEFOREtherapy (N=16), and statistically non-distinguishable AFTER therapy(N=12), P=0.1392. C. The “patient” group (N=27) showed defective pathwayfunction BEFORE therapy, and the defect was corrected AFTER therapy,showing statistically significant on the effect of the treatment,P<0.0001.

FIGS. 6A-6C show results from a CD8+ Treg cell specificity assay as setforth in Example 13. The CD8+ Treg cells from dermatomyositis (DM)patients are functionally defective when compared with the normalfunctional CD8+ Treg cells from healthy individuals prior to treatmentwith the therapeutic agent of the present invention, whereas thefunction of the defective CD8+ Treg cells from the DM patients wascorrected or restored after the treatment. A. BEFORE therapy, thereadings between “normal” (N=16) and “patients” (N=14) are statisticallysignificant, P<0.0001. B. The “control” group showed normal pathwayfunction BEFORE therapy (N=16), and statistically non-distinguishableAFTER therapy (N=12), P=0.1392. C. The “patient” group (N=14) showeddefective pathway function BEFORE therapy, and the defect was correctedAFTER therapy, showing statistically significant on the effect of thetreatment, P<0.0001.

FIGS. 7A-7C show results from a CD8+ Treg cell specificity assay as setforth in Example 14. The CD8+ Treg cells from Pemphigus patients arefunctionally defective when compared with the normal functional CD8+Treg cells from healthy individuals prior to treatment with thetherapeutic agent of the present invention, whereas the function of thedefective CD8+ Treg cells from the DM patients was corrected or restoredafter the treatment. A. BEFORE therapy, the readings between “normal”(N=16) and “patients” (N=18) are statistically significant, P<0.0001. B.The “control” group showed normal pathway function BEFORE therapy(N=16), and statistically non-distinguishable AFTER therapy (N=14),P=0.1392. C. The “patient” group (N=18) showed defective pathwayfunction BEFORE therapy, and the defect was corrected AFTER therapy,showing statistically significant on the effect of the treatment,P<0.0001.

FIGS. 8A-8C show results from a CD8+ Treg cell specificity assay as setforth in Example 15. The CD8+ Treg cells from most of clinicallydiagnosed multiple sclerosis (MS) patients, whom were randomly tested,diagnosed as SPMS (secondary-progressive multiple sclerosis), werefunctionally defective when compared with the normal functional CD8+Treg cells from healthy individuals prior to treatment with thetherapeutic agent of the present invention, whereas the function of thedefective CD8+ Treg cells from the majority of the MS patients testedwas corrected or restored after the treatment. A. BEFORE therapy, thereadings between “normal” (N=16) and “SPMS patients” (N=16) arestatistically significant, P<0.0001. B. The “control” group showednormal pathway function BEFORE therapy (N=16), and statisticallynon-distinguishable AFTER therapy (N=12), P=0.1392. C. The “patient”group (N=16) showed defective pathway function BEFORE therapy, andcorrected defect AFTER therapy, showing statistically significant on theeffect of the treatment, P<0.0001.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to dendritic cells (DCs) prepared from a subject,loaded with human heat shock protein 60 signal peptide (hHsp60sp), andfixed to stabilize the binding between the HLA-E molecules on DCs andthe hHsp60sp. The resulting DCs are referred to pDC(H)s. The inventionalso provides an assay and novel cells lines to determine whether asubject has defective HLA-E restricted CD8+ Treg cells and to determinewhether the defective HLA-E restricted CD8+ Treg cells from a subjectare correctable by treatment with autologous pDC(H)s ex vivo. Theinvention further relates to the use of autologous pDC(H)s to correctdefective HLA-E restricted CD8+ Treg cells in the subject in vivo totreat an autoimmune disease in the subject.

This invention is based on the discovery by the inventor that fixationof the hHsp60sp loaded DCs with 2% paraformaldehyde (PFA) stabilizes theassociation between the binding of HLA-E on DCs and the hHsp60sp andimproves stability of the pDC(H)s from preparation to administration,while the pDC(H)s remain therapeutically effective. The fixation of thepDC(H)s makes it feasible for preparation of the autologous pDC(H)s froma subject, screening for a patient having defective HLA-E restrictedCD8+ Treg cells that are correctable by the autologous pDC(H)s,correcting defective HLA-E restricted CD8+ Treg cells in a subject, andtreating a patient suffering from an autoimmune disease.

The invention is also based on the development of two novel cells lines.These novel cell lines make the potency assay more reliable andefficient to identify a subject having defective HLA-E restricted CD8+Treg cells that are correctable by the autologous pDC(H)s. The two novelcell lines are human HLA-A/B/C—deficient B lymphoma cell lines havingstable surface expression of a complex of HLA-E and the hHsp60sp (TH1)or human HLA-A/B/C— deficient lymphoma B cell lines having stablesurface expression of a complex of HLA-E and a control peptide, forexample, B7sp (TB1). These cell lines are critical to the consistencyand stability of the potency assay.

The invention is further based on the discovery by the inventor thattreatment of patients suffering from type 1 diabetes (T1D) withautologous pDC(H)s resulted in restoration of the CD8 Treg function,stop or delay of the decline of C-peptide AUC, maintaining HbA1c withina normal range, and reducing daily insulin intake up to one year afterthe treatment. Moreover, the inventor has successfully correcteddefective HLA-E restricted CD8+ Treg cells from patients suffering frommultiple sclerosis, psoriasis, rheumatoid arthritis, lupus, vitiligo,pemphigus or dermatomyositis. The correction was performed ex-vivo withautologous pDC(H)s.

Unlike existing small molecule or cell-based therapies, the presentinvention provides for a new type of cell-based therapeutic approach totreat and/or prevent T1D and other autoimmune diseases in patients. Thetherapeutic agent in the present invention, used as a vaccine, may treatthe patients by re-activating and correcting the defective regulatorypathway mediated by HLA-E restricted CD8+ Treg cells, which under normalcircumstances act to selectively control the pathogenic self-reactive Tcells that destroy β cells without damaging the ongoing normal immuneresponses to their anti-infection and anti-tumor immunity, a processcalled self-nonself discrimination.

All terms used in the present application have the plain and ordinarymeanings and unless defined otherwise.

TABLE 2 List of Abbreviations AABB American Association of Blood BanksAPC Antigen Presenting Cells ATG Anti-thymocyte Globulin B721 A HumanHLA-A/B/C-deficient B cell line B721/E B721 cell line transfected withHLA-E gene B7sp Human eguivalent peptide of Qdm; VMAPRTVLL (SEQ ID NO:2) CD11C+ Integrin alpha X chain protein, which is found at a high levelon human DCs CD3+ Cluster of Differentiation 3 positive T cells; T cellco-receptor CD4 Cluster of differentiation 4 CD8 Cluster ofdifferentiation 8 CD8(B) CD8+ T cells stimulated with autologousimmature dendritic cells loaded with B7sp CD8(H) CD8+ T cells stimulatedwith autologous immature dendritic cells loaded with hHsp60sp CD8(N)CD8+ T cells stimulated with autologous immature dendritic cells notloaded with peptide CD8+ T cells Cluster of Differentiation 8 positive Tcells; cytotoxic T cells CD86+ T cells Cluster of Differentiation 86positive T cells CFSE Carboxyfluorescein succinimidyl ester CTCAE CommonTerminology Criteria for Adverse Events CTL Cytotoxic T Cell DO Day 0 D5Day 5 D6 Day 6 D11 Day 11 DC Dendritic cell DLT Dose limiting toxicityDMK Dystrophia myotonica kinase DMSO Dimethyl sulfoxide DSMB Data SafetyMonitoring Board FACS Fluorescence-activated cell sorting FBS Fetalbovine serum GAD Glutamic acid decarboxylase GCP Good clinical practiceGFAP Glial fibrillary acidic protein GM-CSF Granulocyte-macrophagecolony-stimulating factor HbA1c Hemoglobin A1c HBsAg Hepatitis B virussurface antigen HBV Hepatitis B virus HCV Hepatitis C Virus HIV Humanimmunodeficiency virus HLA Human leukocyte antigen HLA-E Human leukocyteantigen (HLA) system E; HLA class I histocompatibility antigen, alphachain E (HLA-E); MHC class 1 antigen E HSA Human serum albumin hHsp60spHuman heat Shock Protein 60 Signal Peptide consisting of QMRPVSRVL (SEQID NO: 1) HTLV-1/2 Human T-lymphotropic virus type 1/2 i.v. IntravenousICA Islet Cell Antibody iDC Immature dendritic cell IFNy Interferon yIGRP Islet-specific glucose-6-phosphate catalytic subunit relatedprotein IL Interleukin IL-2 Interleukin 2 IL-4 Interleukin 4 LAL Limulusamebocyte lysate LN2 Liquid nitrogen mcg Micrograms MHC Majorhistocompatibility complex MMRM Mixed model for repeated measures MMTTMixed meal tolerance test MRI Magnetic resonance image mRNA Messengerribonucleic acid NOD Non-obese diabetic (murine model) PBMC Peripheralblood mononuclear cells PBS Phosphate buffered saline pDC ; fixedpeptide loaded dendritic cell; fixed peptide pulsed dendritic cellpDC(H) Fixed hHsp60sp loaded dendritic cell; Fixed hHsp60sp pulseddendritic cell pDC(B) Fixed B7sp loaded dendritic cell; Fixed B7sppulsed dendritic cell PFA Paraformaldehyde ppIAPP Pre-pre-islet amyloidpolypeptide protein Qa-1 Murine MHC class 1 molecule RA Rheumatoidarthritis RNA ribonucleic acid RPMI Roswell Park Memorial Institutemedium RRMS Relapsing-remitting multiple sclerosis S.C. Subcutaneous SLESystemic lupus erythematosus SPMA Secondary-progressive multiplesclerosis T1D Type 1 diabetes TB T cell line having surface expressionof an HLA-E/B7sp complex TH T cell line having surface expression of anHLA-E/Hsp60sp complex TNC Total nucleated cells TNFα Tumor necrosisfactor α Treg cells Regulatory T cells USP United States Pharmacopeia

(1). The term “type A peptide” used herein refers to a HLA-E bindingpeptide Qdm (in mice) and B7sp (in humans), or Qdm/B7sp like peptidethat can interact with CD94/NKG2A when bound to HLA-E and inhibit thefunction of natural killer (NK) cells.

(2). The term “type B peptide” used herein refers to a HLA-E bindingpeptide having a common structure of x-Met/Leu-x-x-x-x-x-x-leu/Ile (xrepresents any amino acid) (SEQ ID NO: 3) that (i) does not inhibit NKcells when bound to HLA-E; (ii) is recognized by HLA-E restricted CD8+Treg cells when bound to HLA-E; (iii) can compete with type A HLA-Ebinding peptides such as Qdm or B7sp for binding to HLA-E; and (vi)inhibit the target cells that expressing the “complex” of HLA-E coupledwith such peptide(s). Hsp60sp and hHsp60sp are type B peptides.

(3). The term “Qdm” used herein refers to an MHC class Ialeader-sequence derived peptide that binds HLA-E under physiologicalconditions and consists of the amino acid sequence AMAPRTLLL (SEQ ID NO:4)

(4). The term “human heat shock protein 60 signal peptide (hHsp60sp)” asused herein refers to the peptide consisting of QMRPVSRVL (SEQ ID NO: 1)Please double check the sequence.

(5). The term “B7sp” used herein refers to the peptide consisting of theamino acid sequence VMAPRTVLL (SEQ ID NO: 2) Please double check thesequence.

(6). The term “dendritic cells (DCs)” used herein refers to a type ofhuman antigen-presenting cells (also known as accessory cells) thatprocess an antigen and present the processed peptide(s) by MHC/HLAmolecules on the cell surface to T cells of the immune system toactivate the T cells. DCs play an important role in connecting theinnate and the adaptive immune systems. No single cell marker has beenfound to be expressed exclusively on DCs. A combination of the presenceand absence of various cell markers are used to identify the DCs.Markers for DCs include BDCA-1, CD8, CD8alpha, CD11b, CD11c, CD103,CD205 and MHC Class Ia and/or MHC class Ib molecules, including HLA-E,and MHC Class I.

(7). The term “Biomarker” used herein refers to Type B peptide/s coupledwith HLA-E molecules preferentially expressed on all self-reactive Tcells that are specifically recognized by the T cell receptor (TCR) onQ/E CD8+ Treg cells.

(8). The term “immature dendritic cells (iDCs)” used herein refers todendritic cells obtained from a culture of mononuclear cells for no morethan 6 days, which mononuclear cells are isolated from a subject.Markers for iDCs include, CD11c.

(9). The term “pDC(H)” used herein refers to a dendritic cell (DC)loaded with hHsp60sp, for example, ex vivo, and fixed with, for example,paraformaldehyde.

(10). The terms “fixation” and “fixing” as used herein refer to achemical process to stabilize or preserving cells and their subcellularcomponents as close as possible to their original status. One suitablefixative is paraformaldehyde (PFA), for example, 2% PFA.

(11). The terms “peptide pulsing” and “peptide loading” are used hereininterchangeably and refer to treating cells in vitro with a peptide(e.g., hHsp60sp and B7sp) to form a complex of the peptide and amolecule (e.g., HLA-E) expressed on the cell surface. The resultingcells are referred to as peptide pulsed or loaded cells. Where the cellsare DCs, this process is also referred to as “DC pulsing” and theresulting cells are referred to as peptide pulsed DCs or loaded DCs.Where the peptide is a HLA-E binding peptide hHsp60sp, and the cells areDCs, the resulting cells are hHsp60sp pulsed DCs.

(12). The term “subject” used herein refers to a human individual. Thesubject may suffer from an autoimmune disease. The subject may haveHLA-E restricted CD8+ Treg cells capable of specifically recognizingHLA-E/hHsp60sp complex expressed on target cells, and/or down-regulatingself-reactive T cells by specifically recognizing HLA-E/hHsp60sp complexexpressed on target cells. The subject may have defective Q/E CD8+ Tregcells. The subject may have defective HLA-E restricted CD8+ Treg cellsthat are correctable by treating the defective HLA-E restricted CD8+Treg cells with autologous pDC(H)s. The subject may have correctabledefective HLA-E restricted CD8+ Treg cells capable of selectivelysuppressing self-reactive T cells when triggered ex-vivo, or inoculatedinto the subjects in vivo with pDC(H)s.

(13). The term “autoimmune disease” used herein refers to a symptom orpathological condition in a subject, whose immune system attacks thesubject's own cells, tissues or organs. Examples of autoimmune diseasesinclude alopecia areata, ankylosing spondylitis, antiphospholipidsyndrome, autoimmune Addison's disease, autoimmune hemolytic anemia,autoimmune hepatitis, autoimmune inner ear disease, autoimmunelymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura(ATP), Behcet's disease, bullous pemphigoid, cardiomyopathy, celiacsprue-dermatitis, chronic fatigue syndrome immune, deficiency syndrome(CFIDS), chronic inflammatory demyelinating polyneuropathy, cicatricialpemphigoid, cold agglutinin disease, crest syndrome, Crohn's disease,Dego's disease, dermatomyositis, dermatomyositis-juvenile, discoidlupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis,Grave's disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathicpulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IGAnephropathy, juvenile arthritis, Meniere's disease, mixed connectivetissue disease, multiple sclerosis (MS), myasthenia gravis, pemphigusvulgaris, pernicious anemia, ply arteritis nodosa, polychondritis,polyglandular syndromes, polymyalgia rheumatic, polymyositis anddermatomyositis, psoriasis, Raynaud's phenomenon, Reiter's syndrome,rheumatic fever, rheumatoid arthritis, scleroderma, scleroderma,Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus(SLE), Takayasu arteritis, temporal arteritis/giant cell arteritis, type1 diabetes (T1D), ulcerative colitis, uveitis, vasculitis, vitiligo andWegener's granulomatosis. The autoimmune disease may be selected fromthe group consisting of type 1 diabetes (T1D), multiple sclerosis (MS),psoriasis, rheumatoid arthritis, lupus, vitiligo, pemphigus anddermatomyositis. In one embodiment, the autoimmune disease is type 1diabetes (T1D). In another embodiment, the autoimmune disease ismultiple sclerosis (MS).

(14). The term “autologous” used herein refers to the source of a sampleof, for example, cells, obtained or derived from a subject to whom thesample is administered. The autologous sample may be modified ex vivobefore being administered to the subject. Certain desirable cells (e.g.,mononuclear cells) may be collected from blood of a subject, andcultured to obtain dendritic cells (DCs), including immature dendriticcells (iDC), which may be harvested and loaded with a desirable peptide,for example, hHsp60sp or a control peptide (e.g., B7sp). The autologouscells may be administered to the subject from whom the autologous cellsare obtained or derived.

(15). The terms “specifically recognize” used herein refers to specificrecognition of a complex on the cell surface comprised of HLA-E moleculeon a cell associated with a desirable peptide (e.g., hHsp60sp), but notany peptide that is not the desirable peptide, for example, a controlpeptide (e.g., B7sp). The specific association may be direct orindirect.

(16). The term “HLA-E” used herein refers to human leukocyte antigen(HLA) system E, also known as HLA class Ib histocompatibility antigen,alpha chain E (HLA-E), or MHC class Ib antigen E. The HLA-E is a proteinencoded by the HLA-E gene in humans. The amino acid sequences of HLA-Emay be found in NCBI accession nos. CAA05527, CAA40172, BAB63328, andBAF31260.

(17). The terms “HLA-E-restricted CD8+ regulatory T cells”,“HLA-E-restricted CD8+ Treg cells”, “HLA-E-restricted CD8+ T cells”,“HLA-E restricted CD8+ T cells”, “HLA-E restricted regulatory CD8+ Tcells” or “Q/E CD8+ Treg cells” used herein refer to CD8+ T cells thatspecifically recognize a type B peptide (e.g., hHsp60sp) presented by aHLA-E molecule on the surface of target cells. Upon the specificrecognition, the HLA-E restricted CD8+ Treg cells may specificallysuppress, inhibit or down regulate self-reactive T cells in the subject.

The term “defective HLA-E restricted CD8+ Treg cells” used herein refersto HLA-E restricted CD8+ Treg cells incapable of specificallyrecognizing a complex of HLA-E associated with hHsp60sp (HLA-E/hHsp60spcomplex). Defective HLA-E restricted CD8+ Treg cells in a subject areincapable of selectively suppressing self-reactive T cells in thesubject. Whether HLA-E restricted CD8+ Treg cells are defective may bedetermined according to the present invention, for example, using a CD8+T cell specificity assay. HLA-E restricted CD8+ Treg cells having apercentage of maximum inhibition less than 50% of HLA-E restricted CD8+Treg cells from a normal healthy control people may be deemed defective.In one embodiment, HLA-E restricted CD8+ Treg cells having a percentageof maximum inhibition less than 50% of HLA-E restricted CD8+ Treg cellsfrom a normal healthy control people are deemed as defective. The term“correctable defective HLA-E restricted CD8+ Treg cells” used hereinrefers to defective HLA-E restricted CD8+ Treg cells from a subject,which defective HLA-E restricted CD8+ Treg cells became capable ofspecifically recognizing a complex of HLA-E associated with hHsp60sp(HLA-E/hHsp60sp complex) after being treated ex vivo with fixeddendritic cells loaded with the hHsp60sp [pDC(H)s], which dendriticcells are from the same subject. Corrected defective HLA-E restrictedCD8+ Treg cells may be administered to the subject. Corrected defectiveHLA-E restricted CD8+ Treg cells administered to the subject mayselectively suppress self-reactive T cells in the subject. Whetherdefective HLA-E restricted CD8+ Treg cells are correctable may bedetermined according to the present invention, for example, using a CD8+T cell specificity assay. Defective HLA-E restricted CD8+ Treg cellshaving a normalized percentage of maximum inhibition greater than 50% ofHLA-E restricted CD8+ Treg cells from a normal healthy control peoplemay be deemed as correctable. In one embodiment, defective HLA-Erestricted CD8+ Treg cells having a normalized percentage of maximuminhibition greater than 50% of HLA-E restricted CD8+ Treg cells from anormal healthy control people are deemed correctable.

(18). The term “target cells” used herein refers to any cells, includingartificially made cells having surface expression of HLA-E/Type Bpeptide complex (for example, hHsp60sp), which are made for performingthe “HLA-E restricted CD8+ Treg specificity assay” to detect thefunction of the Q/E CD8+ Treg cells.

(19). The term “unloaded target cells” used herein refers to targetcells having surface expression of HLA-E not loaded with a peptide. Forexample, the unloaded target cells may be HLA-E transfected B721 cells(B721/E).

(20). The term “loaded target cells” used herein refers to target cellshaving surface expression of HLA-E loaded with a peptide. Examples ofloaded target cells include B721/E cells loaded with hHsp60sp (specifictarget cells) or B7sp (control target cells).

(21). The terms “specific target cells”, “H cells”, or “TH1 cells” usedherein interchangeably and refers to T cells having surface expressionof HLA-E/hHsp60sp complex. The HLA-E and the hHsp60sp form anHLA-E/hHsp60sp complex on the surface of the specific target cells,which may be cells (e.g., B721) transfected with a gene encoding theHLA-E or a fusion protein of the HLA-E and the hHsp60sp. The HLA-E orthe fusion protein may be expressed transiently or permanently. In oneembodiment, the specific target cell is of a cell line expressing thefusion protein of the HLA-E and the hHsp60sp on the cell surface (ATCCAccession No. PTA-127256).

(22). The terms “control target cells”, “B cells”, or “TB1 cells” usedherein interchangeably and refers to T cells having surface expressionof HLA-E that have been pulsed or loaded with a control peptide (e.g.,B7sp). The HLA-E and the control peptide (e.g., B7sp) form anHLA-E/control peptide complex. The control target cells may be cellstransfected with a gene encoding the HLA-E or a fusion protein of theHLA-E and the control peptide (e.g., B7sp). The HLA-E or the fusionprotein may be expressed transiently or permanently. In one embodiment,the control target cell is of a cell line expressing the fusion proteinof the HLA-E and the B7sp on the cell surface (ATCC Accession No.PTA-127257).

When used in the same assay or method, the specific target cells,control target cells and unloaded target cells may be derived from thesame parental cells, for example, B721 cells, a Human B cell lymphomacell line.

(23). The term “graded ratios” used herein refers to a series of ratiosbetween two components in a mixture designed for changing thecharacteristics of the mixture.

(24). The term “freshly isolated” used herein refers to mononuclearcells that have not been cultured and triggered with pDC after beingisolated from a subject.

(25). The terms “activation” or “activating” used herein refer tostimulation of or stimulating CD8+ regulatory T cells by fixedautologous DCs loaded with a peptide. The CD8+ regulatory T cells areHLA-E-restricted. The HLA-E-restricted CD8+ Treg cells may be defective.The autologous DCs loaded with a peptide may have been purified. Thepeptide may be hHsp60sp or a control peptide (e.g., B7sp). CD8+ T cellsactivated by fixed hHsp60sp loaded autologous DCs [pDC(H)s] are referredto as CD8(H). CD8+ T cells activated by autologous DCs loaded with B7spare referred to as CD8(B).

(26). The terms “HLA-E-restricted regulatory CD8+ T cell function”,“HLA-E-restricted CD8+ T cell function”, “CD8 Treg function” or “CD8+ Tcell function” used herein refers to the ability of the HLA-E-restrictedregulatory CD8+ T cells, HLA-E-restricted CD8+ T cells, CD8 Treg or CD8+T cells, which are capable of specifically recognizing HLA-E/hHsp60spcomplex, to specifically inhibit, suppress or down-regulateproliferation of target cells. Failure to do that indicates that theHLA-E-restricted regulatory CD8+ T cells, HLA-E-restricted CD8+ T cells,CD8 Treg or CD8+ T cells are defective. This defect may be correctable.

(27). The terms “CD8+ T cell specificity assay”, “potency test”,“potency assay” or “CD8+ T cell inhibition assay” used herein refers toan in vitro test to determine the inhibition effect of testing CD8+ Tcells, which are capable of specifically recognizing HLA-E/hHsp60spcomplex, on the surface of target cells. The target cells are incubatedin the presence of testing CD8+ T cells, acting as effector cells (E),at graded E/T ratios (experimental cultures) or in the absence oftesting CD8+ T cells (control cultures). The target cells may be a cellmixture of an equal number of target cells (e.g., specific target cellsexpressing an HLA-E/hHsp60sp complex or control target cells expressingan HLA-E/hB7sp complex) and internal system control target cells(parental B721 cells). The ratio of number of the cultured specifictarget cells vs the number of an internal system control target cells(B721) in the absence of testing CD8+ T cells or in the presence oftesting CD8+ T cells at each of the graded E/T ratios is quantified as acontrol ratio or an experimental ratio, respectively, and used tocalculate a percentage of specific inhibition (potency) for the specifictarget cells or control target cells vs internal system control cells,at each graded E/T ratio.

(28). The term “percentage of specific inhibition (potency)” used hereinrefers to an inhibition measurement for testing CD8+ T cells on eitherspecific target cells or on control target cells in the presence of thegradient number of the testing CD8+ T cells as compared with that oneither specific or control target cells in the absence of testing CD8+ Tcells as follows:

specific inhibition (%)=(control ratio−experimental ratio)/controlratio×100%.

The control ratio is the ratio of the quantified proliferation of themixture of specific target cells or control target cells mixed withinternal system control cells (B721) in the absence of the testing CD8+T cells (well 1). The experimental ratio is the ratio of the quantifiedproliferation of the specific target cells or control target cells mixedwith the internal system control cells (B721) in the presence of thetesting CD8+ T cells at a gradient E/T ratio. The percentage of specificinhibition for the testing CD8 T cells on the target cells may varydepending on the E/T ratio, and may be used to generate an inhibitioncurve and identify the percentage of the maximum inhibition (InhibitionIndex) for the loaded target cells or artificially made transfectants.

(29). The term “inhibition curve” used herein refers to a curvegenerated by the percentages of specific inhibition for each testingCD8+ T cells on each target cells at various E/T ratios. Where thetarget cells are specific target cells such as B721/E loaded with Type Bpeptide such as hHsp60sp peptide, the inhibition curve is a specificinhibition curve and the highest value on the specific inhibition curveis the percentage of the maximum inhibition for the specific targetcells. Where the target cells are control target cells, such as B721/Eloaded with Type A peptide, the inhibition curve is a control inhibitioncurve and the highest value on the control inhibition curve is thepercentage of the maximum inhibition for the control target cells. Thepercentage of the maximum inhibition for the specific target cells andthe percentage of the maximum inhibition for the control target cellsmay be used to calculate an “inhibition index” for testing CD8+ T cells.

(30). The terms “inhibition index”, used herein refer to an inhibitionmeasurement for testing CD8+ T cells on specific target cells ascompared with control target cells, and equals to the percentage of themaximum inhibition for the specific target cells (i.e., highest value ofthe specific inhibition curve) subtracted by the percentage of themaximum inhibition for the control target cells (i.e., the highest valueof the control inhibition curve).

(31). The term “D0-assay” used herein refers to an inhibitionmeasurement for testing CD8+ T cells on specific target cells ascompared with control target cells before activation by autologouspDC(H)s. The D0-assay is to detect the function of the HLA-E restrictedregulatory CD8+ T cells without any treatment with pDC(H)s, such asfreshly isolated CD8+ T cells from the subject/s.

(32). The term “D11-assay” used herein refers to an inhibitionmeasurement for defective CD8+ T cells on specific target cells afterbeing triggered by fixed autologous DCs loaded with hHsp60sp [CD8(H)cells)] as compared with control target cells after being triggered byfixed autologous DCs loaded with a control peptide B7sp [CD8(B) cells].The “Inhibition Index” equals to a percentage of maximum inhibition forthe defective CD8+ T cells after being triggered by the fixed autologousDCs loaded with hHsp60sp subtracted by a percentage of maximuminhibition for the defective CD8+ T cells after being triggered by theautologous DCs loaded with the control peptide B7sp. The D11-assay is todetect the function of the dysfunctional HLA-E restricted regulatoryCD8+ T cells after any treatment(s) with pDC(H)s to judge if thedysfunction of the CD8+ Treg cells tested is correctable.

(33). The term “therapeutic agent”, “therapeutic composition” or“therapeutic preparation” used herein refers to a chemical entity,composition, formulation, preparation, complex, compound or moleculethat exhibits one or more desirable therapeutic properties. Thetherapeutic agent of the present invention is autologous dendritic cellsloaded with hHsp60sp [pDC(H)s].

(34). The term “therapeutically effective amount” used herein refers toan amount of a therapeutic agent effective for achieving a targetedgoal, for example, slowing, halting or reversing the progression of adisease in a subject. The therapeutically effective amount may be anamount required for ameliorating or lessening a symptom, or altering orimproving a biological indicator associated with the disease, forexample, function of the HLA-E restricted CD8+ Treg, or the efficacy ofthe subject with autoimmune diseases by the treatment with pDC(H)s. Thetherapeutically effective amount may depend on nature of the targetedgoal (e.g., correcting defective HLA-E restricted CD8+ regulatory Tcells, treating an autoimmune disease, treating a type 1 diabetes, ortreating multiple sclerosis), the nature of the therapeutic agent, thenature of the therapeutic composition, the subject, and theadministration route. The therapeutically effective amount may bedetermined by a physician.

(35). The term “pharmaceutically acceptable carrier” used herein refersto a pharmaceutically acceptable material, composition or vehicle, forexample, a liquid or solid filler, diluent, excipient, solvent orencapsulating material depending on the route of administration.

(36). The term “administration” or “administering” used herein refers todelivery of, for example, a therapeutic composition of the presentinvention, to a subject via any route, for example, via an intravenous,oral, nasal, transmucosal, transdermal, intramuscular and subcutaneousroute. In one embodiment, the delivery is carried out by intravenousinfusion.

(37). The term “cryoprotectant” used herein refers to a substance thatprevents or minimizes damage to cells during a freezing process. Thecryoprotectant may be selected from the group consisting of glycerol,propylene glycol, dimethyl sulfoxide (DMSO), and a combination thereof.

(38). The term “immunophenotyping” used herein refers to a process thatuses antibodies and/or any other methods to identify certain type ofcells based on one or more unique or specific types of the cell surfaceor intracellular antigens or markers.

(39). The terms “sterile” or “sterility” used herein refers to nodetectable growth of potentially infectious agents such as bacteriaand/or fungi in a testing sample for a predetermined period of time, forexample, at least 14 days in a sterility test. The sterility test may bea commercially available sterility test.

(40). The term “pDC(B)” used herein refers to a dendritic cell (DC)loaded with B7sp, for example, ex vivo, and fixed with, for example,paraformaldehyde.

First, the present invention provides a composition comprising dendriticcells loaded with hHsp60sp. The dendritic cells are from a subject inneed of the dendritic cells loaded with hHsp60sp, and the dendriticcells loaded with hHsp60sp have been fixed with paraformaldehyde (PFA).The fixed dendritic cells loaded with hHsp60sp are also referred to aspDC(H)s. The pDC(H)s may be in a therapeutically effective amount forcorrecting correctable defect of HLA-E restricted CD8+ Treg cells from asubject, for treating an autoimmune disease in a subject.

In one embodiment, the composition is a therapeutic composition forcorrecting defective HLA-E restricted CD8+ Treg cells in vivo from asubject. The defective HLA-E restricted CD8+ Treg cells regain thecapacity of selective suppression of self-reactive T cell pool leadingto the amelioration of the autoimmune disease of the subject aftertriggered (ex-vivo) or treated (in vivo) by the pDC(H)s. The therapeuticcomposition comprises a therapeutically effective amount of autologousdendritic cells loaded with the hHsp60sp [pDC(H)s] in a medium. ThepDC(H)s have been fixed.

The hHsp60sp is the human heat shock protein 60 signal peptideconsisting of the amino acid sequence of QMRPVSRVL (SEQ ID NO: 1). ThehHsp60sp may be in the form of sterile white powder, and may bereconstituted with ddH2O to obtain, for example, a final concentrationof 1-3 mM (e.g., 2 mM) as stock solution.

In the therapeutic composition, the fixed hHsp60sp loaded autologousdendritic cells [pDC(H)s)] is the therapeutic agent. The autologouspDC(H)s are obtained from a subject from whom the defective HLA-Erestricted CD8+ Treg cells are obtained. The autologous DCs may beisolated from the same subject or derived from cells isolated from thesubject. The autologous DCs may be DCs harvested from a culture ofmononuclear cells from the subject for a predetermined period of time,for example, 3, 4, 5, 6, 7 or 8 days. Where the autologous DCs harvestedfrom a culture of mononuclear cells from the subject for no more than 6days, the harvested DCs are immature DCs (iDCs).

The autologous DCs loaded with the hHsp60sp [pDC(H)s] are autologous DCspulsed with the hHsp60sp forming a complex between the hHsp60sp andHLA-E expressed on the surface of the autologous DCs. Stable structureof the complex between the association of hHsp60sp and HLA-E moleculeson the autologous DCs is critical to the stability and therapeuticeffect of the pDC(H)s.

To improve stability of the pDC(H)s, the pDC(H)s have been fixed tostabilize the structure of the complex of HLA-E/hHsp60sp on the cellsurface. The pDC(H)s may be fixed by a fixative that causes covalentcross-links between the hHsp60sp and the HLA-E to keep them together asa complex. The pDC(H)s may be fixed by paraformaldehyde (PFA), forexample, 2% PFA.

The therapeutically effective amount of the autologous pDC(H)s is anamount of the autologous pDC(H)s required for correcting defective HLA-Erestricted CD8+ Treg cells from a subject. The therapeutically effectiveamount may be determined by a physician.

In one embodiment of the present invention, a patient who has beendiagnosed with having defective HLA-E restricted CD8+ Treg cells may beadministered intravenously a treatment effective amount of the fixedhHsp60sp peptide-loaded dendritic cells (pDC(H)s) in a range betweenabout 0.3×10⁶ and about 90×10⁶ cells, preferably about 2×10⁶ and about20×10⁶ cells, most preferably about 7×10⁶ and about 10×10⁶ cells, in asuspension comprising pharmaceutically acceptable carriers.

In another embodiment, a patient who has been diagnosed with diagnosedwith having defective HLA-E restricted CD8+ Treg cells may beadministered intravenously a treatment effective amount of the fixedhHsp60sp peptide-loaded dendritic cells [pDC(H)s] in a range betweenabout 0.3×10⁶ and about 90×10⁶ cells, preferably about 2×10⁶ and about20×10⁶ cells, most preferably about 7×10⁶ and about 10×10⁶ cells, in asuspension comprising pharmaceutically acceptable carriers, two or moretimes separated by at least 14 days, preferably by 21 days.

In a further embodiment, a patient who has been diagnosed with diagnosedwith having defective HLA-E restricted CD8+ Treg cells may beadministered intravenously a treatment effective amount of the fixedhHsp60sp peptide-loaded dendritic cells [pDC(H)s] in a range betweenabout between about 0.3×10⁶ and about 90×10⁶ cells, preferably about2×10⁶ and about 20×10⁶ cells, most preferably about 7×10⁶ and about10×10⁶ cells, in a volume between 10 ml and 50 ml, preferably between 15ml and 30 ml, of a suspension comprising pharmaceutically acceptablecarriers, two or more times separated by at least 14 days, preferably byat least 21 days.

In a further embodiment, a patient who has been diagnosed with diagnosedwith having defective HLA-E restricted CD8+ Treg cells may beadministered intravenously a treatment effective amount of the fixedhHsp60sp peptide-loaded dendritic cells [pDC(H)s) in a range betweenabout 0.3×10⁶ and about 90×10⁶ cells, preferably between about 2×10⁶ andabout 20×10⁶ cells, most preferably between about 7×10⁶ and about 10×10⁶cells, in a volume between 10 ml and 50 ml, preferably between 15 ml and30 ml, of a suspension pharmaceutically acceptable carriers, two or moretimes separated by 30 (+/−7) days.

The medium may comprise dimethyl sulfoxide (DMSO), human serum albumin(HAS) and plasmalyte-A. For example, the medium may include 2 mLdimethyl sulfoxide (DMSO), 10 mL 25% human serum albumin (HAS) and 8 mLPlasmalyte-A.

The therapeutic composition may comprise a pharmaceutically acceptablecarrier. The pharmaceutically acceptable carrier may be apharmaceutically acceptable material, composition or vehicle, forexample, a liquid or solid filler, diluent, excipient, solvent orencapsulating material depending on the route of administration.

The therapeutic composition comprises cells expressing DC markers.Suitable DC markers include CD11C and CD3. The therapeutic compositionmay comprise greater than 60%, 70%, 80% or 90% total CD11c+(gated onlarge cells). For example, the therapeutic composition may comprisegreater than 99.0-99.9% total CD11c+(gated on large cells).

The therapeutic composition may be sterile. The sterility may bedetermined based on absence of detectable growth of bacteria,mycoplasma, and/or fungi in a testing sample for a predetermined periodof time, for example, at least 14 days.

The therapeutic composition may further comprise a cryoprotectant. Thecryoprotectant may be any substance that prevents or minimizes thedamage to cells or tissues during freezing process and its subsequentstorage period. The cryoprotectant may be selected from the groupconsisting of glycerol, propylene glycol, dimethyl sulfoxide (DMSO), anda combination thereof.

The therapeutic composition may be frozen. The therapeutic compositionmay be frozen by a conventional method. The therapeutic composition maybe frozen in a controlled rate freezer, and then stored in vapor phaseliquid nitrogen before use.

In one embodiment, the therapeutic composition is formulated forintravenous administration to the subject.

The subject may be a human individual. The subject may suffer from anautoimmune disease. The autoimmune disease may be selected from thegroup consisting of alopecia areata, ankylosing spondylitis,antiphospholipid syndrome, autoimmune Addison's disease, autoimmunehemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease,autoimmune lymphoproliferative syndrome (ALPS), autoimmunethrombocytopenic purpura (ATP), Behcet's disease, bullous pemphigoid,cardiomyopathy, celiac sprue-dermatitis, chronic fatigue syndromeimmune, deficiency syndrome (CFIDS), chronic inflammatory demyelinatingpolyneuropathy, cicatricial pemphigoid, cold agglutinin disease, crestsyndrome, Crohn's disease, Dego's disease, dermatomyositis,dermatomyositis-juvenile, discoid lupus, essential mixedcryoglobulinemia, fibromyalgia-fibromyositis, Grave's disease,Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis,idiopathic thrombocytopenia purpura (ITP), IGA nephropathy, juvenilearthritis, Meniere's disease, mixed connective tissue disease, multiplesclerosis (MS), myasthenia gravis, pemphigus vulgaris, perniciousanemia, ply arteritis nodosa, polychondritis, polyglandular syndromes,polymyalgia rheumatic, polymyositis and dermatomyositis, psoriasis,Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoidarthritis, scleroderma, scleroderma, Sjogren's syndrome, stiff-mansyndrome, systemic lupus erythematosus (SLE), Takayasu arteritis,temporal arteritis/giant cell arteritis, type 1 diabetes (T1D),ulcerative colitis, uveitis, vasculitis, vitiligo and Wegener'sgranulomatosis. In one embodiment, the autoimmune disease may be type 1diabetes (T1D), multiple sclerosis (MS), psoriasis, lupus vitiligo,pemphigus or dermatomyositis. In another embodiment, the autoimmunedisease is type 1 diabetes (T1D). In yet another embodiment, theautoimmune disease is multiple sclerosis (MS).

Second, the present invention provides a method for preparing acomposition. The method comprises isolating mononuclear cells from asubject; incubating the mononuclear cells in a culture to producedendritic cells (DCs); harvesting the DCs from the culture; incubatingthe harvested DCs with hHsp60sp to produce DCs loaded with the hHsp60sp(hHsp60sp loaded DCs); fixing the hHsp60sp loaded DCs to produced fixedhHsp60sp loaded DCs [pDC(H)s]; and suspending the pDC(H)s in a medium.As a result, the composition is prepared.

The mononuclear cells may be isolated from the subject by a conventionalmethod (e.g., Leukapheresis). The isolated mononuclear cells may beincubated in a culture under conditions inducing production of DCs. Themononuclear cells may be incubated in the presence of one or morecytokines, for example, GM-CSF and IL-4.

The isolated mononuclear cells may be incubated in the culture for nomore than 4-6 days, preferably no more than 6 days, to produce DCs. TheDCs harvested from the mononuclear cells are incubated in the culturefor no more than 6 days are immature DCs (iDCs). The iDCs may becharacterized by a marker such as CD11c when harvested.

The harvested DCs may have a cell count of at least 2×10⁷, 3×10⁷, 4×10⁷,5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷ or 10×10⁷.

The harvested DCs comprise cells expressing DC markers. The harvestedDCs may comprise greater than 60%, 70%, 80% or 90% total CD11c+(gated onlarge cells). For example, the harvested DCs may comprise greater than99.0-99.9% total CD11c+(gated on large cells).

The harvested DCs may be sterile. The sterility may be determined basedon absence of detectable growth of bacteria, mycoplasma and/or fungi ina testing sample for a predetermined period of time, for example, atleast 14 days.

In the peptide pulsing step, the harvested DCs (e.g., iDCs) areincubated with the hHsp60sp under conditions permitting association ofthe harvested DCs with the hHsp60sp. At least 3×10⁷, 4×10⁷, 5×10⁷,6×10⁷, 7×10⁷ or 8×10⁷ harvested DCs may be incubated with the hHsp60sp.For example, a minimum of 5×10⁷ DCs may be peptide pulsed with thehHsp60sp at 100-150 μM for 2 hours at 37° C., 5% CO₂ and 60-90% RelativeHumidity (RH).

In the fixing step, the hHsp60sp loaded DCs may be fixed by a fixativethat causes covalent cross-links between the hHsp60sp and HLA-E to keepthem together in the complex. The hHsp60sp loaded DCs may be fixed byparaformaldehyde (PFA), for example, 2% PFA. The fixed hHsp60sp loadedDCs are also referred to as pDC(H)s.

In the suspending step, the pDC(H)s may be mixed with a medium of 1-100mL, 5-50 mL, 10-50 mL, 15-30 mL, 18-22 mL or 20 mL. The medium maycomprise dimethyl sulfoxide (DMSO), human serum albumin (HAS) andplasmalyte-A. In one embodiment, the medium includes 2 mL dimethylsulfoxide (DMSO), 10 mL 25% human serum albumin (HAS) and 8 mLPlasmalyte-A.

The prepared composition may comprise a pharmaceutically acceptablecarrier. The pharmaceutically acceptable carrier may be apharmaceutically acceptable material, composition or vehicle, forexample, a liquid or solid filler, diluent, excipient, solvent orencapsulating material depending on the route of administration. Thepharmaceutically acceptable carrier does not reduce the therapeuticeffect of the pDC(H)s by, for example, more than 50%, 40%, 30%, 20%, 10%or 5%.

The prepared composition may comprise a cryoprotectant. Thecryoprotectant may be any substance that prevents or minimizes damage tothe pDC(H)s during the freezing process. The cryoprotectant may beselected from the group consisting of glycerol, propylene glycol,dimethyl sulfoxide (DMSO), and a combination thereof.

The preparation method may further comprise freezing the preparedcomposition. The prepared composition may be frozen by a conventionalmethod. The prepared composition may be frozen in a controlled ratefreezer, and then stored in vapor phase liquid nitrogen before use. Thefrozen pDC may remain effective at least 24 months.

The prepared composition may be formulated for administration to thesubject via any route, for example, via an intravenous, oral, nasal,transmucosal, transdermal, intramuscular and subcutaneous route. In oneembodiment, the prepared composition is formulated for intravenousadministration to the subject

According to the preparation method, the subject may be a humanindividual. The subject may suffer from an autoimmune disease,preferably the autoimmune disease is diagnosed within three months toone year from the present treatment. A shorter period between diagnosisof an autoimmune disease and treatment is mostly preferred. Theautoimmune disease may be selected from the group consisting of alopeciaareata, ankylosing spondylitis, antiphospholipid syndrome, autoimmuneAddison's disease, autoimmune hemolytic anemia, autoimmune hepatitis,autoimmune inner ear disease, autoimmune lymphoproliferative syndrome(ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease,bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronicfatigue syndrome immune, deficiency syndrome (CFIDS), chronicinflammatory demyelinating polyneuropathy, cicatricial pemphigoid, coldagglutinin disease, crest syndrome, Crohn's disease, Dego's disease,dermatomyositis, dermatomyositis-juvenile, discoid lupus, essentialmixed cryoglobulinemia, fibromyalgia-fibromyositis, Grave's disease,Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis,idiopathic thrombocytopenia purpura (ITP), IGA nephropathy, juvenilearthritis, Meniere's disease, mixed connective tissue disease, multiplesclerosis (MS), myasthenia gravis, pemphigus vulgaris, perniciousanemia, ply arteritis nodosa, polychondritis, polyglandular syndromes,polymyalgia rheumatic, polymyositis and dermatomyositis, psoriasis,Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoidarthritis, scleroderma, scleroderma, Sjogren's syndrome, stiff-mansyndrome, systemic lupus erythematosus (SLE), Takayasu arteritis,temporal arteritis/giant cell arteritis, type 1 diabetes (T1D),ulcerative colitis, uveitis, vasculitis, vitiligo and Wegener'sgranulomatosis. In one embodiment, the autoimmune disease may be type 1diabetes (T1D), multiple sclerosis (MS), psoriasis, rheumatoidarthritis, lupus, vitiligo, pemphigus or dermatomyositis. In anotherembodiment, the autoimmune disease is type 1 diabetes (T1D). In yetanother embodiment, the autoimmune disease is multiple sclerosis (MS).

The pDC(H)s prepared according to the preparation method of the presentinvention may be used in the therapeutic composition of the presentinvention.

Third, the present invention provides recombinant human cells.

A recombinant human cell comprising a heterologous gene encoding afusion protein is provided. The recombinant cell expresses the fusionprotein on the surface of the cell. The fusion protein comprises humanleukocyte antigen system E (HLA-E) and hHsp60sp. The fusion protein mayfurther comprise a linker between the HLA-E and the hHsp60sp. The linkermay be 10-20 amino acids long. A Gly-Ser linker may be used. Therecombinant human cell may also comprise a GFP protein or the like as anindicator. The recombinant cell may express the fusion protein and GFPprotein permanently. The recombinant human cell is a cell line having anATCC accession number of PTA-127256. [HLA-E/hHsp60sp]

A recombinant human cell comprising a heterologous gene encoding afusion protein is provided. The recombinant cell expresses the fusionprotein on the surface of the cell. The fusion protein comprises humanleukocyte antigen system E (HLA-E) and B7sp. The fusion protein mayfurther comprise a linker between the HLA-E and the B7sp. The linker maybe 10-20 amino acids long. A Gly-Ser linker may be used. The recombinanthuman cell may also comprise a GFP protein or the like as an indicator.The recombinant cell may express the fusion protein and GFP proteinpermanently. The recombinant human cell is a cell line having an ATCCaccession number of PTA-127257. [HLA-E/B7sp]

Fourth, the present invention provides a HLA-E restricted CD8+ Tregspecificity assay, also referred to as a potency assay. Testing HLA-Erestricted CD8+ Treg cells from a subject may be characterized by itspercentage of maximum inhibition in a potency assay (Inhibition Index).The testing HLA-E restricted CD8+ Treg cells are capable of specificallyrecognizing HLA-E/hHsp60sp complex (the biomarker) expressed on thetarget cells. The percentage of inhibition of the testing HLA-Erestricted CD8+ Treg cells may be calculated based on the specificinhibition of specific target cells compared with control target cells,to test the function of HLA-E restricted CD8+ Treg cells in a PotencyAssay. The percentage of inhibition for testing HLA-E restricted CD8+Treg cells from a subject may be used to determine whether a subject hasdefective HLA-E restricted CD8+ Treg cells and whether defective HLA-Erestricted CD8+ Treg cells from a subject are correctable. Here, thecontrol target cells are cells expressing a complex of HLA-E and acontrol type A peptide, such as B7sp. The specific target cells arecells expressing a complex of HLA-E and Type B peptide, such ashHsp60sp. The specific target cells, the control target cells and theinternal control cells are derived from the same parental cell line(e.g., B721).

In the Potency Assay, in each testing group of CD8+ T cells, specificand control target cells are plated into two separate rows in a 48-wellplate and incubated in the absence of the CD8+ T cells (well 1 in eachrow), or in the presence of the testing CD8+ T cells (e.g. experimentalwells 2-6 in each row). In both rows, the CD8+ T cells were acting aseffector cells (E), at graded E/T ratios, for example, from 3:1 to0.005:1. The inhibition curve will be calculated in both rows of agroups.

The specific target cells may comprise a first heterologous geneencoding a first fusion protein of the HLA-E and the hHsp60sp andexpress the first fusion protein on the surface of the specific targetcells. The first fusion protein may further comprise a linker betweenthe HLA-E and the hHsp60sp. The linker may be 10-20 amino acids long. AGly-Ser linker may be used. The recombinant human cell may also comprisea GFP protein or the like as an indicator. The specific target cells mayexpress the first fusion protein and GFP protein permanently. Thespecific target cells may be cells of the cell line having an ATCCaccession number of PTA-127256.

The control target cells may comprise a second heterologous geneencoding a second fusion protein of HLA-E and B7sp and express thesecond fusion protein on the surface of the control target cells. Thesecond fusion protein may further comprise a linker between the HLA-Eand the B7sp. The linker may be 10-20 amino acids long. A Gly-Ser linkermay be used. The recombinant human cell may also comprise a GFP proteinor the like as an indicator. The control target cells may express thesecond fusion protein and GFP protein permanently. The control targetcell may be of a cell line having an ATCC accession number ofPTA-127257.

Proliferation of either specific target cells (hHsp60sp loaded B721/Emixed with B721 or TH1 mixed with B721) or control target cells (B7sploaded B721/E mixed with B721 or TB1 mixed with B721) in the absence oftesting CD8+ T cells (Control culture, well 1 of the row) or in thepresence of testing CD8+ T cells (Experimental culture, well 2-6 of therow) at each of the grading E/T ratios is quantified. At each E/T ratio,a percentage of specific inhibition for the specific or control targetcells is calculated based on the quantified proliferation of theExperimental ratio—Control ratio as follows:

Specific inhibition (%) of either specific targets or controltargets=(Experimental ratio−Control ratio)/Control ratio×100%.

The control ratio is the ratio of the quantified proliferation of thecontrol target cells or the specific target cells in the absence of CD8+T cells. The Experimental ratio is the quantified proliferation ratio ofcontrol target cells or the specific target cells in the presence of thegradient amount of testing CD8+ T cells. An inhibition curve may begenerated to show percentages of specific inhibition for the specifictarget cells and control targets at different E/T ratios.

The percentage of maximum inhibition (Inhibition Index) for the testingHLA-E restricted CD8+ Treg cells equals to the percentage of the maximuminhibition for the specific target cells subtracted by the percentage ofthe maximum inhibition for the control target cells. The value of theInhibition Index for the testing HLA-E restricted CD8+ Treg cells may beused to determine whether the testing HLA-E restricted CD8+ Treg cellsare defective in a D0 assay and correctable in a D11 assay.

A method is provided to determine a percentage of maximum inhibition(Inhibition Index) for testing the function of the HLA-E restricted CD8+Treg cells from a subject. The read out is the specific inhibition ofthe testing HLA-E restricted CD8+ Treg cells to suppress the specifictarget cells via a specific recognition of HLA-E/hHsp60sp complex (thebiomarker) expressed on the target cells. This assay is to test if theHLA-E restricted CD8+ Treg cells are capable of specifically recognizingthe “biomarker” (HLA-E/hHsp60sp complex) expressed on the target cells.The method comprising (a) determining inhibition for specific targetcells; (b) determining inhibition for control target cells; and (c)calculating maximum inhibition (Inhibition Index) of the testing HLA-Erestricted CD8+ Treg cells 4-from each subject by subtracting thepercentage of maximum inhibition for the control target cells from thepercentage of maximum inhibition for the specific target cells. Thespecific target cells are cells expressing HLA-E/hHsp60sp complex (thebiomarker) on the surface of the target cells. The control target cellsare target cells expressing the HLA-E/control peptide complex (controlfor the biomarker) on the surface of the control target cells. Thespecific target cells may be the cell line having an ATCC accessionnumber of PTA-127256 while the control target cells may be the cell linehaving an ATCC accession umber of PTA-127257.

According to the method of determining an “Inhibition Index” of testingHLA-E restricted CD8+ Treg cells from a subject, steps comprise (i)obtaining a specific target cell mixture having an equal number ofspecific or control target cells with the internal control target cell,e.g. B721; (ii) incubating the specific vs control target cell mixturein the absence or presence of the gradient testing HLA-E restricted CD8+regulatory T cells in two separate rows; (iii) quantifying proliferationof the specific target cells as well as the control target cells tocalculate the specific target cell mixture in the presence or absence ofthe testing HLA-E restricted CD8+ Treg cell at graded specific E/Tratios and (vi) calculating a percentage of maximum specific inhibitionfor the specific target cells (Inhibition Index) by picking up thehighest % of inhibition of the “inhibition curve” of the specifictargets, subtracted by the highest % of inhibition of the controltargets.

According to the method of determining a percentage of maximuminhibition for testing HLA-E restricted CD8+ Treg cells from a subject,the specific target cells may be from the cell line having an ATCCaccession number of PTA-127256 while the control target cells may befrom the cell line having an ATCC accession number of PTA-127257.

According to the method of determining a percentage of maximuminhibition for testing HLA-E restricted CD8+ Treg cells from a subject,the subject may be a human individual. The subject may suffer anautoimmune disease. The autoimmune disease may be selected from thegroup consisting of alopecia areata, ankylosing spondylitis,antiphospholipid syndrome, autoimmune Addison's disease, autoimmunehemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease,autoimmune lymphoproliferative syndrome (ALPS), autoimmunethrombocytopenic purpura (ATP), Behcet's disease, bullous pemphigoid,cardiomyopathy, celiac sprue-dermatitis, chronic fatigue syndromeimmune, deficiency syndrome (CFIDS), chronic inflammatory demyelinatingpolyneuropathy, cicatricial pemphigoid, cold agglutinin disease, crestsyndrome, Crohn's disease, Dego's disease, dermatomyositis,dermatomyositis-juvenile, discoid lupus, essential mixedcryoglobulinemia, fibromyalgia-fibromyositis, Grave's disease,Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis,idiopathic thrombocytopenia purpura (ITP), IGA nephropathy, juvenilearthritis, Meniere's disease, mixed connective tissue disease, multiplesclerosis (MS), myasthenia gravis, pemphigus vulgaris, perniciousanemia, ply arteritis nodosa, polychondritis, polyglandular syndromes,polymyalgia rheumatic, polymyositis and dermatomyositis, psoriasis,Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoidarthritis, scleroderma, scleroderma, Sjogren's syndrome, stiff-mansyndrome, systemic lupus erythematosus (SLE), Takayasu arteritis,temporal arteritis/giant cell arteritis, type 1 diabetes (T1D),ulcerative colitis, uveitis, vasculitis, vitiligo and Wegener'sgranulomatosis. In one embodiment, the autoimmune disease may be type 1diabetes (T1D), multiple sclerosis (MS), psoriasis, rheumatoidarthritis, lupus, vitiligo, pemphigus or dermatomyositis. In anotherembodiment, the autoimmune disease is type 1 diabetes (T1D). In yetanother embodiment, the autoimmune disease is multiple sclerosis (MS).

A method is provided to determine whether HLA-E restricted CD8+ Tregcells freshly isolated from a subject are defective (D0 assay). Thefreshly isolated HLA-E restricted CD8+ Treg cells from normal healthyindividuals are capable of selectively suppressing self-reactive T cellsby specifically recognizing HLA-E/hHsp60sp complex expressed on thespecific target cells. The method comprises determining a percentage ofmaximum inhibition (Inhibition Index) for the freshly isolated HLA-Erestricted CD8+ Treg cells according to the method described above usingthe freshly isolated HLA-E restricted CD8+ Treg cells as the testingHLA-E restricted CD8+ Treg cells. In one embodiment, a percentage ofmaximum inhibition from a testing subject is less than 50% of HLA-Erestricted CD8+ T cells freshly isolated from a normal healthy controlsubject indicates that the HLA-E restricted CD8+ Treg cells freshlyisolated from the testing subject are defective, that is, the subjecthas defective HLA-E restricted CD8+ Treg cells.

According to the method of determining whether HLA-E restricted CD8+Treg cells from a subject are defective, the specific target cells maybe from the cell line having an ATCC accession number of PTA-127256while the control target cells may be from the cell line having an ATCCaccession number of PTA-127257.

According to the method of determining whether HLA-E restricted CD8+Treg cells from a subject are defective, the subject may be a humanindividual. The subject may suffer an autoimmune disease. The autoimmunedisease may be selected from the group consisting of alopecia areata,ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison'sdisease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmuneinner ear disease, autoimmune lymphoproliferative syndrome (ALPS),autoimmune thrombocytopenic purpura (ATP), Behcet's disease, bullouspemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatiguesyndrome immune, deficiency syndrome (CFIDS), chronic inflammatorydemyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinindisease, crest syndrome, Crohn's disease, Dego's disease,dermatomyositis, dermatomyositis-juvenile, discoid lupus, essentialmixed cryoglobulinemia, fibromyalgia-fibromyositis, Grave's disease,Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis,idiopathic thrombocytopenia purpura (ITP), IGA nephropathy, juvenilearthritis, Meniere's disease, mixed connective tissue disease, multiplesclerosis (MS), myasthenia gravis, pemphigus vulgaris, perniciousanemia, ply arteritis nodosa, polychondritis, polyglandular syndromes,polymyalgia rheumatic, polymyositis and dermatomyositis, psoriasis,Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoidarthritis, scleroderma, scleroderma, Sjogren's syndrome, stiff-mansyndrome, systemic lupus erythematosus (SLE), Takayasu arteritis,temporal arteritis/giant cell arteritis, type 1 diabetes (T1D),ulcerative colitis, uveitis, vasculitis, vitiligo and Wegener'sgranulomatosis. In one embodiment, the autoimmune disease may be type 1diabetes (T1D), multiple sclerosis (MS), psoriasis, rheumatoidarthritis, lupus, vitiligo, pemphigus or dermatomyositis. In anotherembodiment, the autoimmune disease is type 1 diabetes (T1D). In yetanother embodiment, the autoimmune disease is multiple sclerosis (MS).

A method is provided to determine whether defective HLA-E restrictedCD8+ Treg cells from a subject are correctable (D11 Assay). The freshlyisolated HLA-E restricted CD8+ Treg cells from normal healthyindividuals or corrected dysfunctional HLA-E restricted CD8+ Treg cellsare capable of selectively suppressing self-reactive T cells byspecifically recognizing the complex of HLA-E/hHsp60sp expressed on thespecific target cells. The defective HLA-E restricted CD8+ Treg cellsare not capable of specifically recognizing complex of HLA-E/hHsp60spexpressed on the target cells. To test if the dysfunctional HLA-Erestricted CD8+ Treg cells are correctable, the method (D11 assay)comprises: (a) triggering the defective HLA-E restricted CD8+ Treg cellswith fixed autologous dendritic cells loaded with hHsp60 [pDC(H)s] toproduce CD8(H) cells ex vivo; (b) determining a percentage of maximuminhibition for the CD8(H) cells according to the method descried aboveusing the CD8(H) cells as the testing HLA-E restricted CD8+ regulatory Tcells; (c) Triggering the defective HLA-E restricted CD8+ Treg cellswith fixed autologous dendritic cells loaded with a control peptide[pDC(B)s] to produce CD8(B) cells ex vivo; (d) determining a percentageof maximum inhibition for the CD8(B) cells according to the method asdescribed above using the CD8(B) cells as control of the testing HLA-Erestricted CD8+ Treg cells; and (e) calculating a maximum inhibition(Inhibition Index) for the treated defective HLA-E restricted CD8+ Tregcells by subtracting the percentage of highest inhibition for the CD8(B)cells from the percentage of highest inhibition for the CD8(H) cells. Inone embodiment, a percentage of maximum inhibition (Inhibition Index)for defective HLA-E restricted CD8+ Treg cells from a subject afterex-vivo trigger is greater than 50% of the normal healthy controlsubject tested that is built in within the same test indicates that thedefective HLA-E restricted CD8+ Treg cells from the subject arecorrectable, that is, the subject has correctable defective HLA-Erestricted CD8+ Treg cells.

According to the method of determining whether the defective HLA-Erestricted CD8+ Treg cells from a subject are correctable, the defectiveHLA-E restricted CD8+ Treg cells are determined according to the methoddescribed above.

According to the method of determining whether the defective HLA-Erestricted CD8+T regulatory cells from a subject are correctable, wherethe defective HLA-E restricted CD8+ Treg cells from the subject aredetermined to be correctable, the method may further comprise correctingthe defective HLA-E restricted CD8+ T cells by autologous pDC(H)s exvivo and administering the pDC(H)s to the subject.

According to the method of determining whether the defective HLA-Erestricted CD8+T regulatory cells from a subject are correctable, thespecific target cells may be of the cell line having an ATCC accessionnumber of PTA-127256 (TH1) while the control target cells may be of thecell line having an ATCC accession number of PTA-127257 (TB1).

According to the method of determining whether the defective HLA-Erestricted CD8+ Treg cells from a subject are correctable, the subjectis a human individual. The subject may suffer from an autoimmunedisease. The autoimmune disease may be selected from the groupconsisting of alopecia areata, ankylosing spondylitis, antiphospholipidsyndrome, autoimmune Addison's disease, autoimmune hemolytic anemia,autoimmune hepatitis, autoimmune inner ear disease, autoimmunelymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura(ATP), Behcet's disease, bullous pemphigoid, cardiomyopathy, celiacsprue-dermatitis, chronic fatigue syndrome immune, deficiency syndrome(CFIDS), chronic inflammatory demyelinating polyneuropathy, cicatricialpemphigoid, cold agglutinin disease, crest syndrome, Crohn's disease,Dego's disease, dermatomyositis, dermatomyositis-juvenile, discoidlupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis,Grave's disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathicpulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IGAnephropathy, juvenile arthritis, Meniere's disease, mixed connectivetissue disease, multiple sclerosis (MS), myasthenia gravis, pemphigusvulgaris, pernicious anemia, ply arteritis nodosa, polychondritis,polyglandular syndromes, polymyalgia rheumatic, polymyositis anddermatomyositis, psoriasis, Raynaud's phenomenon, Reiter's syndrome,rheumatic fever, rheumatoid arthritis, scleroderma, scleroderma,Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus(SLE), Takayasu arteritis, temporal arteritis/giant cell arteritis, type1 diabetes (T1D), ulcerative colitis, uveitis, vasculitis, vitiligo andWegener's granulomatosis. In one embodiment, the autoimmune disease maybe type 1 diabetes (T1D), multiple sclerosis (MS), psoriasis, rheumatoidarthritis, lupus, vitiligo, pemphigus or dermatomyositis. In anotherembodiment, the autoimmune disease is type 1 diabetes (T1D). In yetanother embodiment, the autoimmune disease is multiple sclerosis (MS).

Fifth, the present invention provides a method for correcting defectiveHLA-E restricted CD8+ Treg cells in a subject in need thereof. Thedefective HLA-E restricted CD8+ Treg cells are correctable and capableof selectively down-regulating self-reactive T cells by specificallyrecognizing a complex of HLA-E/hHsp60sp expressed on the targetself-reactive T cells in vivo after administrated with pDC(H)s which iscapable of activating the HLA-E restricted CD8+ Treg cells, in vivo, tocorrect their function. The method comprises administering to thesubject a therapeutically effective amount of a therapeutic composition.The therapeutic composition comprises fixed autologous dendritic cellsloaded with the hHsp60sp (pDC(H)s) in a medium. The pDC(H)s have beenfixed with paraformaldehyde (PFA).

According to the method of correcting defective HLA-E restricted CD8+Treg cells in a subject in need thereof, whether a subject has defectiveHLA-E restricted CD8+ Treg cells may be determined by the assaydescribed above based on the Inhibition Index for HLA-E restricted CD8+Treg cells freshly isolated from the subject (D0 assay) followed by aD11 assay to test if the defective CD8+ Tregs can be corrected byex-vivo retrigger with pDC(H)s, according to the present invention.

According to the method of correcting defective HLA-E restricted CD8+Treg cells in a subject in need thereof, whether defective HLA-Erestricted CD8+ Treg cells from a subject are correctable may bedetermined based on the Inhibition Index for the defective HLA-Erestricted CD8+ Treg cells from the subject before (D0 assay) and afteractivation (D11 assay) by autologous pDC(H)s according to the presentinvention. The autologous pDC(H)s may be prepared according to thepresent invention. The autologous pDC(H)s may be in a therapeuticcomposition according to the present invention.

According to the correction method, the pDC(H)s may be fixed by afixative that causes covalent cross-links between the hHsp60sp and HLA-Eto keep them together in the complex and attached to an insolublenetwork without reducing the therapeutic effect of the pDC(H)s by, forexample, more than 50%, 40%, 30%, 20%, 10% or 5%. The pDC(H)s may befixed by paraformaldehyde (PFA), for example, 2% PFA.

Patients diagnosed with having defective HLA-E restricted CD8+ Tregcells may be treated with the therapeutic agent in the present inventionby intravenous administration of the therapeutic agent at a dosingregimen determined by a medical professional.

In one embodiment of the present invention, a patient who has beendiagnosed with having defective HLA-E restricted CD8+ Treg cells may beadministered intravenously a treatment effective amount of the fixedhHsp60sp peptide-loaded dendritic cells [pDC(H)s) in a range betweenabout 0.3×10⁶ and about 90×10⁶ cells, preferably about 2×10⁶ and about20×10⁶ cells, most preferably about 7×10⁶ and about 10×10⁶ cells, in asuspension comprising pharmaceutically acceptable carriers.

In another embodiment, a patient who has been diagnosed with havingdefective HLA-E restricted CD8+ Treg cells may be administeredintravenously a treatment effective amount of the fixed hHsp60sppeptide-loaded dendritic cells [pDC(H)s] in a range between about0.3×10⁶ and about 90×10⁶ cells, preferably about 2×10⁶ and about 20×10⁶cells, most preferably about 7×10⁶ and about 10×10⁶ cells, in asuspension comprising pharmaceutically acceptable carriers, two or moretimes separated by at least 14 days, preferably by at least 21 days.

In a further embodiment, a patient who has been diagnosed with havingdefective HLA-E restricted CD8+ Treg cells may be administeredintravenously a treatment effective amount of the fixed hHsp60sppeptide-loaded dendritic cells [pDC(H)s] in a range between aboutbetween about 0.3×10⁶ and about 90×10⁶ cells, preferably about 2×10⁶ andabout 20×10⁶ cells, most preferably about 7×10⁶ and about 10×10⁶ cells,in a volume between 10 ml and 50 ml, preferably between 15 ml and 30 ml,of a suspension comprising pharmaceutically acceptable carriers, two ormore times separated by at least 14 days, preferably by at least 21days.

In a further embodiment, a patient who has been diagnosed with havingdefective HLA-E restricted CD8+ Treg cells may be administeredintravenously a treatment effective amount of the fixed hHsp60sppeptide-loaded dendritic cells [pDC(H)s] in a range between about0.3×10⁶ and about 90×10⁶ cells, preferably between about 2×10⁶ and about20×10⁶ cells, most preferably between about 7×10⁶ and about 10×10⁶cells, in a volume between 10 ml and 50 ml, preferably between 15 ml and30 ml, of a suspension pharmaceutically acceptable carriers, two or moretimes separated by 30 (+/−7) days.

According to the correction method, the medium may comprise dimethylsulfoxide (DMSO), human serum albumin (HAS) and plasmalyte-A. Forexample, the medium may include 2 mL dimethyl sulfoxide (DMSO), 10 mL25% human serum albumin (HAS) and 8 mL Plasmalyte-A.

According to the correction method, the therapeutic composition maycomprise a pharmaceutically acceptable carrier. The pharmaceuticallyacceptable carrier may be a pharmaceutically acceptable material,composition or vehicle, for example, a liquid or solid filler, diluent,excipient, solvent or encapsulating material depending on the route ofadministration. The pharmaceutically acceptable carrier does not reducethe therapeutic effect of the pDC(H)s by, for example, more than 50%,40%, 30%, 20%, 10% or 5%.

According to the correction method, the therapeutic compositioncomprises cells expressing DC markers. Suitable DC markers include CD11Cand CD3. The therapeutic composition may comprise greater than 60%, 70%,80% or 90% total CD11c+(gated on large cells). For example, thetherapeutic composition may comprise greater than 99.0-99.9% totalCD11c+(gated on large cells).

According to the correction method, the therapeutic composition may besterile. The sterility may be determined based on absence of detectablegrowth of bacteria and/or fungi in a testing sample for a predeterminedperiod of time, for example, at least 14 days.

According to the correction method, the therapeutic composition mayfurther comprise a cryoprotectant. The cryoprotectant may be anysubstance that prevents or minimizing freezing of tissues or damage tocells during freezing. The cryoprotectant may be selected from the groupconsisting of glycerol, propylene glycol, dimethyl sulfoxide (DMSO), anda combination thereof.

According to the correction method, the therapeutic composition may befrozen. The therapeutic composition may be frozen by a conventionalmethod. The therapeutic composition may be frozen in a controlled ratefreezer, and then stored in vapor phase liquid nitrogen before use.

According to the correction method, the therapeutic composition may beformulated for administration to the subject via any route, for example,via an intravenous, oral, nasal, transmucosal, transdermal,intramuscular and subcutaneous route. In one embodiment, the therapeuticcomposition is formulated for intravenous administration to the subject.

According to the correction method, the subject may be a humanindividual. The subject may suffer an autoimmune disease. The autoimmunedisease may be selected from the group consisting of alopecia areata,ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison'sdisease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmuneinner ear disease, autoimmune lymphoproliferative syndrome (ALPS),autoimmune thrombocytopenic purpura (ATP), Behcet's disease, bullouspemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatiguesyndrome immune, deficiency syndrome (CFIDS), chronic inflammatorydemyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinindisease, crest syndrome, Crohn's disease, Dego's disease,dermatomyositis, dermatomyositis-juvenile, discoid lupus, essentialmixed cryoglobulinemia, fibromyalgia-fibromyositis, Grave's disease,Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis,idiopathic thrombocytopenia purpura (ITP), IGA nephropathy, juvenilearthritis, Meniere's disease, mixed connective tissue disease, multiplesclerosis (MS), myasthenia gravis, pemphigus vulgaris, perniciousanemia, ply arteritis nodosa, polychondritis, polyglandular syndromes,polymyalgia rheumatic, polymyositis and dermatomyositis, psoriasis,Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoidarthritis, scleroderma, scleroderma, Sjogren's syndrome, stiff-mansyndrome, systemic lupus erythematosus (SLE), Takayasu arteritis,temporal arteritis/giant cell arteritis, type 1 diabetes (T1D),ulcerative colitis, uveitis, vasculitis, vitiligo and Wegener'sgranulomatosis. In one embodiment, the autoimmune disease may be type 1diabetes (T1D), multiple sclerosis (MS), psoriasis, rheumatoidarthritis, lupus, vitiligo, pemphigus or dermatomyositis.

Sixth, the present invention provides a method for treating type 1diabetes (T1D) in a subject in need thereof. The T1D treatment methodcomprises administering to the subject a therapeutically effectiveamount of a therapeutic composition. The therapeutic compositioncomprises autologous dendritic cells loaded with the hHsp60sp [pDC(H)s]in a medium. The pDC(H)s have been fixed with paraformaldehyde (PFA).

In the T1D treatment method, for being used as a therapy the autologouspDC(H)s may be prepared according to the present invention. Thetherapeutic composition is of the present invention.

According to the T1D treatment method, the hHsp60sp loaded DCs may befixed by a fixative to produce fixed hHsp60sp loaded DCs [pDC(H)s]. Thefixative causes covalent cross-links between the hHsp60sp and HLA-E tokeep them together as the complex without reducing the therapeuticeffect of the pDC(H)s by, for example, more than 50%, 40%, 30%, 20%, 10%or 5% as compared with the hHsp60sp loaded DCs. The hHsp60sp loaded DCsmay be fixed by paraformaldehyde (PFA), for example, 2% PFA.

Patients diagnosed with autoimmune disorders or diseases such as T1D maybe treated with the therapeutic agent in the present invention byintravenous administration of the therapeutic agent at a dosing regimendetermined by a medical professional.

In one embodiment of the present invention, a patient who has beendiagnosed with T1D may be administered intravenously a treatmenteffective amount of the fixed hHsp60sp peptide-loaded dendritic cells[pDC(H)s] in a range between about 0.3×10⁶ and about 90×10⁶ cells,preferably about 2×10⁶ and about 20×10⁶ cells, most preferably about7×10⁶ and about 10×10⁶ cells, in a suspension comprisingpharmaceutically acceptable carriers.

In another embodiment, a patient who has been diagnosed with T1D may beadministered intravenously a treatment effective amount of the fixedhHsp60sp peptide-loaded dendritic cells [pDC(H)s] in a range betweenabout 0.3×10⁶ and about 90×10⁶ cells, preferably about 2×10⁶ and about20×10⁶ cells, most preferably about 7×10⁶ and about 10×10⁶ cells, in asuspension comprising pharmaceutically acceptable carriers, two or moretimes separated by at least 14 days, preferably by at least 21 days.

In a further embodiment, a patient who has been diagnosed with T1D maybe administered intravenously a treatment effective amount of the fixedhHsp60sp peptide-loaded dendritic cells [pDC(H)s] in a range betweenabout between about 0.3×10⁶ and about 90×10⁶ cells, preferably about2×10⁶ and about 20×10⁶ cells, most preferably about 7×10⁶ and about10×10⁶ cells, in a volume between 10 ml and 50 ml, preferably between 15ml and 30 ml, of a suspension comprising pharmaceutically acceptablecarriers, two or more times separated by at least 14 days, preferably byat least 21 days.

In a further embodiment, a patient who has been diagnosed with T1D maybe administered intravenously a treatment effective amount of the fixedhHsp60sp peptide-loaded dendritic cells [pDC(H)s] in a range betweenabout 0.3×10⁶ and about 90×10⁶ cells, preferably between about 2×10⁶ andabout 20×10⁶ cells, most preferably between about 7×10⁶ and about 10×10⁶cells, in a volume between 10 ml and 50 ml, preferably between 15 ml and30 ml, of a suspension pharmaceutically acceptable carriers, two or moretimes separated by 30 (+/−7) days.

T1D may be diagnosed in accordance with any medically acceptablestandards by a competent medical professional or a person skilled in theart, preferably, the diagnosis may be confirmed by positive lab resultfor one or more of the autoantibodies glutamic acid decarboxylase(GAD65), insulinoma associated protein 2 (IA-2, also known as ICA-512)and Zinc transporter 8 (ZnT8).

According to the method of treating T1D patients with presenttherapeutic composition, determination whether a T1D patient hasdefective HLA-E restricted regulatory CD8+ T cells or pathway prior tothe treatment is not required so long as the patient has been diagnosedby a conventional diagnostic method practiced by a person ordinaryskilled in the art.

As understood by a person skilled in the art, all dosing regimensdescribed herein may be modified or adjusted depending on an individualpatient' physical conditions and needs when treating the patient. Thoseembodiments may be used singularly, in any combination, or in anyarrangement to the extent within the skill and knowledge of a personskilled in the art.

According to the T1D treatment methods, the medium may comprise dimethylsulfoxide (DMSO), human serum albumin (HSA) and plasmalyte-A. Forexample, the medium may include 2 mL dimethyl sulfoxide (DMSO), 10 mL25% human serum albumin (HAS) and 8 mL Plasmalyte-A.

According to the T1D treatment method, the therapeutic composition maycomprise a pharmaceutically acceptable carrier. The pharmaceuticallyacceptable carrier may be a pharmaceutically acceptable material,composition or vehicle, for example, a liquid or solid filler, diluent,excipient, solvent or encapsulating material depending on the route ofadministration. The pharmaceutically acceptable carrier does not reducethe therapeutic effect of the pDC(H)s by, for example, more than 50%,40%, 30%, 20%, 10% or 5%.

According to the T1D treatment method, the therapeutic compositioncomprises cells expressing DC markers. Suitable DC markers include CD11Cand CD3. The therapeutic composition may comprise greater than 60%, 70%,80% or 90% total CD11c+(gated on large cells). For example, thetherapeutic composition may comprise greater than 99.0-99.9% totalCD11c+(gated on large cells).

According to the T1D treatment method, the therapeutic composition maybe sterile. The sterility may be determined based on absence ofdetectable growth of bacteria and/or fungi in a testing sample for apredetermined period of time, for example, at least 14 days.

According to the T1D treatment method, the therapeutic composition mayfurther comprise a cryoprotectant. The cryoprotectant may be anysubstance that prevents or minimizing damage to cells during freezingprocess. The cryoprotectant may be selected from the group consisting ofglycerol, propylene glycol, dimethyl sulfoxide (DMSO), and a combinationthereof.

According to the T1D treatment method, the therapeutic composition maybe frozen. The therapeutic composition may be frozen by a conventionalmethod. The therapeutic composition may be frozen in a controlled ratefreezer, and then stored in vapor phase liquid nitrogen before use.

According to the T1D treatment method, the therapeutic composition maybe formulated for administration to the subject via any route, forexample, via an intravenous, oral, nasal, transmucosal, transdermal,intramuscular and subcutaneous route. In one embodiment, the therapeuticcomposition is formulated for intravenous administration to the subject.

According to the T1D treatment method, the therapeutic composition maybe administered to the subject at least twice separated by at least 14days, or by at least three times separated by at least 21 days.

According to the T1D treatment method, patients receiving the presenttherapeutic composition may be treated within 12 months after the T1Ddiagnosis, preferably within three months after the diagnosis. Earliertreatment after T1D diagnosis is beneficial to patients who receive thetreatment of the present therapeutic composition. Patients receiving thepresent treatment may be a minor.

Seventh, the present invention provides a method for treating multiplesclerosis (MS) in a subject in need thereof. The MS treatment methodcomprises administering to the subject a therapeutically effectiveamount of a therapeutic composition. The therapeutic compositioncomprises autologous dendritic cells loaded with the hHsp60sp [pDC(H)s]in a medium. The pDC(H)s have been fixed with paraformaldehyde (PFA).

In the MS treatment method, the autologous pDC(H)s may be preparedaccording to the present invention. The therapeutic composition is ofthe present invention.

According to the MS treatment method, the pDC(H)s may be fixed by afixative that causes covalent cross-links between the hHsp60sp and HLA-Eto keep them together in the complex and attached to an insolublenetwork without reducing the therapeutic effect of the pDC(H)s by, forexample, 2% PFA.

Patients diagnosed with autoimmune disorders or diseases such as MS maybe treated with the therapeutic agent in the present invention byintravenous administration of the therapeutic agent at a dosing regimendetermined by a medical professional.

In one embodiment of the present invention, a patient who has beendiagnosed with MS may be administered intravenously a treatmenteffective amount of the fixed hHsp60sp peptide-loaded dendritic cells[pDC(H)s] in a range between about 0.3×10⁶ and about 90×10⁶ cells,preferably about 2×10⁶ and about 20×10⁶ cells, most preferably about7×10⁶ and about 10×10⁶ cells, in a suspension comprisingpharmaceutically acceptable carriers.

In another embodiment, a patient who has been diagnosed with MS may beadministered intravenously a treatment effective amount of the fixedhHsp60sp peptide-loaded dendritic cells [pDC(H)s] in a range betweenabout 0.3×10⁶ and about 90×10⁶ cells, preferably about 2×10⁶ and about20×10⁶ cells, most preferably about 7×10⁶ and about 10×10⁶ cells, in asuspension comprising pharmaceutically acceptable carriers, two or moretimes separated by at least 14 days, preferably by at least 21 days.

In a further embodiment, a patient who has been diagnosed with MS may beadministered intravenously a treatment effective amount of the fixedhHsp60sp peptide-loaded dendritic cells [pDC(H)s] in a range betweenabout between about 0.3×10⁶ and about 90×10⁶ cells, preferably about2×10⁶ and about 20×10⁶ cells, most preferably about 7×10⁶ and about10×10⁶ cells, in a volume between 10 ml and 50 ml, preferably between 15ml and 30 ml, of a suspension comprising pharmaceutically acceptablecarriers, two or more times separated by at least 14 days, preferably byat least 21 days.

In a further embodiment, a patient who has been diagnosed with MS may beadministered intravenously a treatment effective amount of the fixedhHsp60sp peptide-loaded dendritic cells [pDC(H)s] in a range betweenabout 0.3×10⁶ and about 90×10⁶ cells, preferably between about 2×10⁶ andabout 20×10⁶ cells, most preferably between about 7×10⁶ and about 10×10⁶cells, in a volume between 10 ml and 50 ml, preferably between 15 ml and30 ml, of a suspension pharmaceutically acceptable carriers, two or moretimes separated by 30 (+/−7) days.

According to the MS treatment methods, the medium may comprise dimethylsulfoxide (DMSO), human serum albumin (HAS) and plasmalyte-A. Forexample, the medium may include 2 mL dimethyl sulfoxide (DMSO), 10 mL25% human serum albumin (HAS) and 8 mL Plasmalyte-A.

According to the MS treatment method, the therapeutic composition maycomprise a pharmaceutically acceptable carrier. The pharmaceuticallyacceptable carrier may be a pharmaceutically acceptable material,composition or vehicle, for example, a liquid or solid filler, diluent,excipient, solvent or encapsulating material depending on the route ofadministration. The pharmaceutically acceptable carrier does not reducethe therapeutic effect of the pDC(H)s by, for example, more than 50%,40%, 30%, 20%, 10% or 5%.

According to the MS treatment method, the therapeutic compositioncomprises cells expressing DC markers. Suitable markers to detect theharvested CDs include CD11C and CD3. The therapeutic composition maycomprise greater than 60%, 70%, 80% or 90% total CD11c+(gated on largecells). For example, the therapeutic composition may comprise greaterthan 99.0-99.9% total CD11c+(gated on large cells).

According to the MS treatment method, the therapeutic composition may besterile. The sterility may be determined based on absence of detectablegrowth of bacteria and/or fungi in a testing sample for a predeterminedperiod of time, for example, at least 14 days.

According to the MS treatment method, the therapeutic composition mayfurther comprise a cryoprotectant. The cryoprotectant may be anysubstance that prevents or minimizing damage to cells during freezingprocess. The cryoprotectant may be selected from the group consisting ofglycerol, propylene glycol, dimethyl sulfoxide (DMSO), and a combinationthereof.

According to the MS treatment method, the therapeutic composition may befrozen. The therapeutic composition may be frozen by a conventionalmethod. The therapeutic composition may be frozen in a controlled ratefreezer, and then stored in vapor phase liquid nitrogen before use.

According to the MS treatment method, the therapeutic composition may beformulated for administration to the subject via any route, for example,via an intravenous, oral, nasal, transmucosal, transdermal,intramuscular and subcutaneous route. In one embodiment, the therapeuticcomposition is formulated for intravenous administration to the subject.

According to the MS treatment method, the therapeutic composition may beadministered to the subject at least twice separated by at least 14days, or by at least three times separated by at least 21 days.

According to the MS treatment method, determination whether a MS patienthas defective HLA-E restricted regulatory CD8+ T cells or pathway priorto the treatment is not required so long as the patient has beendiagnosed by a conventional diagnostic method practiced by a personordinary skilled in the art.

Example 1. Manufacture of Fixed hHsp60sp Loaded Autologous DendriticCells [pDC(H)s]

Autologous dendritic cells loaded with a hHsp60sp were manufactured bycollecting mononuclear cells from a subject (e.g., patient) byleukapheresis collection and cell separation/enrichment, differentiatingthe collected primary mononuclear cells into dendritic cells (DCs) incell cultures in the presence of GM-CFS and IL-4, harvesting immatureDCs (iDCs), loading the harvested iDCs with the hHsp60sp (SEQ ID NO: 1),and fixing hHsp60sp loaded DCs to stabilize the association of HLA-E andthe hHsp60sp on the DCs before being fixed to produce pDC(H)s. ThepDC(H)s may be a Therapeutic Agent for administration to the subject.

Key reagents and excipients used in manufacturing the pDC(H)s, includingtheir sources and grades, are shown in Table 1.

TABLE 1 Reagents and essential supplies used in manufacture of pDC(H)sReagent/ Use Concentration Source Grade Excipient at Use Ficoll-PaqueDensity Gradient N/A GE GMP Premium Cell Genix DC Media Culture N/A CellGenix GMP Phosphate Buffered In process washing, IX Gibco or ResearchSaline (PBS) and dilution of PFA equivalent RMPI media Dilution and inN/A Lonza or Research process washing equivalent Fetal Boven serum Tomake the 10% Biotechne complete medium GM-CSF Culture additive 80 ng/mLGenzyme USP IL-4 Culture additive 20 ng/mL Miltenyi Biotec Research orequivalent Hsp60sP Peptide Peptide pulsing 100-150 μM Polypeptide GMPGroup AG Paraformaldehyde Preparation of THE 2% Electron ResearchSolution THERAPEUTIC Microscopy AGENT Sciences 25% Human Serum Washesand N/A Grifols or USP Albumin (25% HSA) Cryopreservation equivalentDimethyl Sulfoxide Cryopreservation 10% Bioniche or GMP (DMSO)equivalent USP for sterility and endotoxin Plasmalyte-A CryopreservationN/A Baxter USP Distilled Water Dilution of peptide N/A Gibco or Researchand Preparation of equivalent stock solution Water for InjectionPreparation of stock N/A Hospira USP solution of cytokines

The hHsp60sp is the human heat shock protein 60 signal peptideconsisting of the amino acid sequence of SEQ ID NO: 1, having amolecular weight (MW) of 1086.33 and in the form of white powder. ThehHsp60sp was reconstituted with ddH2O to obtain a final concentration of2 mM. Aliquots of the reconstituted hHsp60sp is filtered and aliquotedin 400 μL were stored at ≤−130° C. prior to use.

A. Mononuclear Cells

Peripheral blood mononuclear cells (PBMCs) were obtained from a subjectthrough apheresis of therapeutic cells (TC-apheresis). Approximately 15L blood from the subject was processed on the Fenwal Amicus CellSeparator, CaridianBCT Spectra Optia, or equivalent system to obtain aleukapheresis product containing white blood cells at about 1-2×10¹⁰.

In the next morning after the leukapheresis product was obtained (Day0), a small portion (e.g., 1-3 mL from 200 mL) was removed from theleukapheresis product for initial quality control (QC) testing,including total nucleated cells (TNC) using automated cell count, Trypanviability, sterility using Bac-T Alert and QC vials. The leukapheresisproduct was diluted in RPMI and then subject to density gradientperformed using Ficoll-Paque Premium so that mononuclear cells werecollected. The collected mononuclear cells were washed several times andthen subject to pre-culture QC testing, including TNC using automatedcell count and Trypan viability.

B. Dendritic Cells (DCs)

On Day 0, the collected mononuclear cells were seeded into flasks, forexample, Triple Flasks or TC Flasks, and incubated at 37° C. for 1.5 to2 hours to allow monocytes to adhere to the bottom of the flasks.Non-adherent monocytes were decanted while the adherent monocytes werewashed several times with PBS to remove as many non-adherent cells aspossible. Cell Genex DC Media with cytokines IL-4 at 20 ng/mL and GM-CSFat 80 ng/mL was added into the flasks to induce differentiation of theadherent monocytes into dendritic cells (DCs), and the cells in theflasks were incubated at 37° C., 5% CO₂ and 60-90% relative humidity(RH).

On Day 2-3 and Day 4-5, approximately half the culture medium in theflasks was removed and replaced with fresh DC media with cytokines IL-4at 20 ng/mL and GM-CSF at 80 ng/mL to induce differentiation of theadherent monocytes into dendritic cells (DCs), and the cells in theflasks were incubated at 37° C., 5% CO₂ and 60-90% RH. As a result,immature DCs (iDCs) were obtained.

C. Dendritic Cells Loaded with hHsp60sp (pDC(H)s)

On Day 5 or D6, the DCs in suspension were harvested in Gibco RoswellPark Memorial Institute (RPMI). The flasks were washed extensively withRPMI to harvest all of the DCs. The harvested DCs were centrifuged andresuspended. The harvested and resuspended cells were DCs and subject toQC testing, including sterility, manual cell count, viability (Trypan),immunophenotyping, QC vials and mycoplasma.

After resuspension, the harvested DCs were pulsed with the hHsp60sp. Aminimum of 4-5×10⁷ harvested DCs were incubated with the hHsp60sppeptide at 100-150 μM for 2 hours at 37° C., 5% CO₂ and 60-90% RH tomake hHsp60sp peptide pulsed DCs, also referred to as DCs loaded withthe hHsp60sp peptide. The hHsp60 peptide pulsed DCs were washed 1× withPBS before being fixed with a 2% Paraformaldehyde solution for 10minutes at 4° C. to make pDC(H)s. Following fixation, the pDC(H)s werewashed 3× with PBS supplemented with human serum albumin (HSA) tooptimize cell recovery. The pDC(H)s may be used as a Therapeutic Agentto treat the subject who is a patient.

After the washes, samples of the pDC(H)s were removed for QC testing,including Manual Cell Count, immunophenotyping, endotoxin (supernatant)and functional assays.

For each patient, 3-5 20-mL bags of pDC(H)s were prepared. Each bagcontains about 1-1.2×10⁷ pDC(H)s in a medium, which included 2 mL DMSO,10 mL of 25% HSA and 8 mL of Plasmalyte-A. After the addition ofcryoprotectant, sterility testing of the pDC(H)s was performed. ThepDC(H)s in the bags were frozen in a controlled rate freezer, and thenstored in vapor phase liquid nitrogen (LN₂) until release testing wascomplete.

The DC percentage in the total harvested cell population varies amongpatients. In a small group of patients, the CD11c+ cells in the totalharvested cell population may be lower than 20%. Under thesecircumstances, the amount of harvested DCs to be pulsed and fixed may bedetermined on a case-by-case basis to satisfy the required cell numberin the final product.

Because the association between the DCs and the hHsp60sp peptide isunstable, the inventor has surprisingly discovered that fixing thehHsp60sp peptide-loaded dendritic cells, for example, by 2%paraformaldehyde (PFA), stabilizes the complex of HLA-E/hHsp60sp on thesurface of the DCs. The DCs may also be fixed by other conventionalmethods known to a person skilled in the art, for example,cross-linking. It is desirable to fix the DCs as soon as possible afterthe wash to stabilize the association between the HLA-E on DCs and thehHsp60sp peptide. It is preferably to fix the DCs no more than 25minutes after the wash, and more preferably no more than about 10-15minutes after the wash.

Example 2. In-Process Testing

Various tests may be conducted during the process for making the pDC(H)sas described in Example 1. Table 2 lists some in-process tests, expectedresults and testing methods.

TABLE 2 In-process tests Stage Test Expected Result Method Initial 14Day Sterility No Growth Bac-T Alert Cell Counts >1 × 10¹⁰ AutomatedPre-Culture Cell Counts >1 × 10¹⁰ Automated (post Ficoll) Harvested CellCount >3 × 10⁷ Manual iDC Immunophenotyping >70% Flow Cytometry TotalCD11C+ (gated on large cells) Immunophenotyping Record result FlowCytometry Total CD3+ (gated on total cells) 14 Day Sterility No GrowthBac-T Alert Therapeutic Potency Assay *(−) or (+) agentImmunophenotyping Record result Flow Cytometry Total CD3+ (gated ontotal cells) *A percentage of maximum inhibition (Inhibition Index) froma testing subject less than 50% of HLA-E restricted CD8+ T cells freshlyisolated from a normal healthy control subject indicates that the HLA-Erestricted CD8+ T cells freshly isolated from the testing subject aredefective, that is, the subject has defective HLA-E restricted CD8+ Tregcells, and vice versa.

A. Sterility (Bacterial and Fungal Testing)

Sterility testing may be performed on the bioMérieux's BacT/Alert® 3DMicrobial Detection System. This method has been validated against the21 CFR 610.12 Millipore Steritest filtration methods. The BacT/Alertsystem is found to have a quicker time to detection at the samesensitivity level. Samples are directly inoculated into the BacT culturebottles (AST and NST). The bottles are then loaded into the BacT/Alert®3D analyzer, and incubated for 14-days at a temperature range between30-35° C. If microorganisms are present in bottles, they will producecarbon dioxide as they metabolize substrates in the media. This carbondioxide changes sensors at the bottom of the bottles from blue-green toyellow. Using this sensor and reflected light, the BacT/Alert monitorsthe production of carbon dioxide and signals users if it determinesbottles to be positive. If, at the end of 14-day incubation, the systemdoes not determine bottles to be positive, it will report them asnegative. If the system detects CO2 production, it will report thatbottle as positive. If a positive is detected during the 14-dayincubation phase, the bottle will be off loaded and a sample sent foridentification and sensitivities. Samples for confirmatory testing (gramstain, identification and sensitivities) are sent to a qualifiedmicrobiology lab.

B. Endotoxin

Endotoxins are wall constituents of gram-negative bacteria that areliberated during bacterial growth and bacterial death. As such,detection of endotoxin is an indirect measurement of the current orrecent presence of bacteria in the cell culture or associated reagentsand supplies. Endotoxin in the final formulation cryopreserved productsmay be detected using the Endosafe PTS™. It is a handheldspectrophotometer that utilizes FDA-licensed LAL disposable cartridges.

C. Mycoplasma

Mycoplasma testing may be performed by an approved vendor laboratory(WuXi AppTec) using an approved Polymerase Chain Reaction kit (GLP30645).

D. Immunophenotyping

The expression of cell surface DC biomarkers may be monitored byfluorescence-activated cell sorting (FACS) analysis to ensure theimmature DC (iDC) phenotype. Both the post-harvest products (e.g., theharvested DCs) and final products (e.g., pDC(H)s) were measured forsurface expression of CD11c. The final product must contain ≥80% CD11cgated on the DC population (large cells). T-Cell marker (CD3) wasanalyzed on the total cell population and the expected result should be≤50%, but this is not a release criterion.

E. “Potency Assay”—HLA-E Restricted CD8+ Treg Specificity Assay

The “Potency Assay” provides a method for determining if testing HLA-Erestricted CD8+ T cells, which specifically recognize or bind thecomplex of HLA-E coupled with hHsp60sp peptide (SEQ ID NO: 1), have adefect in inhibiting proliferation of specific target cells expressingHLA-E associated with the hHsp60sp peptide (“H cells”) as compared withthat of control target cells expressing on their surface a false markersuch as HLA-E associated with B7sp (SEQ ID NO: 2) (“B cells”). Thismethod may be used 1) to identify a patient who has defective HLA-Erestricted CD8+ T cells and determine if the defect can be corrected; 2)to evaluate potency or function of the pDC(H)s as a therapeutic agent tocorrect the defect and treat any disease or disorder caused by thedefect; and/or 3) to monitor efficacy of the therapeutic agent after thetreatment.

Where the testing HLA-E restricted CD8+ T cells are freshly isolatedfrom a subject (e.g., patient) and fail to inhibit the growth of the Hcells as compared with the B cells, the testing HLA-E restricted CD8+ Tcells are deemed to have a defect and the subject is deemed to need acorrection of such defect. For example, in a D0 assay of the PotencyAssay, the “Inhibition Index”, a difference between the percentage ofthe maximum inhibition of the H cells and the percentage of the maximuminhibition of the B cells, that is less than 50% of the “InhibitionIndex” of a sample that is from a healthy normal subject built in thesame test as a Normal Control, indicates that the testing HLA-Erestricted CD8+ T cells from the subject have a defect.

Where the defective testing HLA-E restricted CD8+ T cells inhibits the Hcells as compared with the B cells after being activated by the pDC(H)s,tested in a D11 assay of the Potency Assay, indicating that 1) thepatients' defective HLA-E restricted CD8+ Treg cells are correctable and2). The pDC(H)s are deemed potent for correcting the defect of thetesting HLA-E restricted CD8+ T cells and may be used as a therapeuticagent for treating a disease or condition caused by the defectivetesting HLA-E restricted CD8+ T cells in the subject. For example, inD11 assay, a net value, i.e., a difference between the percentage of themaximum inhibition of the H cells and the percentage of the maximuminhibition of the B cells (the “Inhibition Index”), that is greater than50% of the Inhibition Index of a sample that is from a healthy normalsubject built in the same test as a Normal Control, indicates that thedefective testing HLA-E restricted CD8+ T cells can be corrected by thepotent pDC(H)s.

1. Protocol

In general, the potency assay includes four steps.

First, target cells are plated. The two types of target cells arespecific target cells expressing the complex of HLA-E associated withthe hHsp60sp peptide (“H cells”) and control target cells expressing ontheir surface a false marker such as the complex of HLA-E associatedwith the B7sp peptide (“B cells”). A system control of parental cellline B721 are mixed with either the specific or control target cells at1:1 ratio to generate a specific or control target cell mixture. The twotarget cell mixtures are each plated into six wells of a 48 well plateat an equal amount.

Second, the testing CD8+ T cells are added into five of the six wells ofeach row in a 48-plate plate (well 2-6) to contact with either thespecific or control target cell mixture in a limiting dilution fashion,leaving the number one well (well 1) without adding the testing T cellsto establish an “inhibition curve” of the specific or control targetcells for the assessment of the function of the testing CD8+ T cells.

Third, the plates are incubated at 37° C., 5% CO₂ for 5-7 days.

Fourth, the readout is the suppression or inhibition of the testing CD8+T cells on proliferation of the specific target cells vs that on thecontrol target cells. An “inhibition curve” for the testing CD8+ T cellsis established on both specific and control target cells by calculatingthe number of the target cells after incubation for 5-7 days with thetesting T cells. An “inhibition index” for the testing CD8+ T cells isdetermined by the difference between inhibition highest values of thespecific inhibition curve and the highest value of the controlinhibition curve.

2. Potency assay (D11 assay) to test 1). if the defective HLA-Erestricted CD8+ Treg cells triggered by pDC(H)s regained the normalfunction or 2). If the pDC(H)s manufactured has the capacity to activatethe defective HLA-E restricted CD8+ Treg cells.

The potency of the pDC(H)s is evaluated by testing if the defectivetesting CD8+ T cells regain the normal function of CD8+ T cellstriggered by the pDC(H)s.

Two testing CD8+ T cell lines CD8(H) and CD8(B) may be established. TheCD8(H) line was generated by co-culturing 0.4-0.5M×10⁶ of purifiedautologous CD8+ T cells with 0.1-0.35×10⁶ pDC(H)s in 1 mL/well in a 48well plate, and thus activated by pDC. The CD8(B) line was generated byco-culturing 0.4-0.5M×10⁶ of purified autologous CD8+ T cells with0.1-0.35×10⁶ DCs loaded with the peptide B7sp peptide in 1 mL/well in a48 well plate, and thus serves as a negative control for CD8(H). IL-2was added on the second day. CD8(H) cells and CD8(B) cells wereharvested on D5 for a potency assay. In each potency assay, the CD8(H)or CD8(B) cells were tested for their effect on proliferation of targetcells.

There are two types of target cells that were prepared and used:

1). The first type of targets: hHsp60sp loaded B721/E cells mixed with*CFSE (carboxyfluorescein succinimidyl ester) labeled B721 cells(specific target cell mixture) vs B7sp loaded B721/E cells mixed with*CFSE (carboxyfluorescein succinimidyl ester) labeled B721 cells(control target cell mixture).

*Here the hHsp60sp or B7sp loaded B721/E cells could be separated fromCFSE labeled B721 cells by the Facs analysis.

2) The second type of targets:

**TH1 cells (expressing HLA-E/hHsp60sp complex and GFP protein on thesurface) mixed with unlabeled B721 as specific target cell mixture; and**TB1 cells (expressing HLA-E/B7sp complex and GFP protein on thesurface) mixed with unlabeled B721 as control target cell mixture.

** Here the GPF protein on TH1 and TB1 cells could separate these twocell lines from the unlabeled B721 by the Facs analysis.

A graded number of CD8(H) and CD8(B) cells were plated into a 48-wellplate in two separate rows as the E/T ratio from 3:1 to 0.01:1 (well 2-6on each row).

An equal number of the specific targets (hHsp60sp loaded B721/E or TH1cells) and control targets (B7sp loaded B721/E or TB1 cells) are mixedwith either CFSE labeled B721 cells (the first type target cells) orunlabeled B721 cells (the second type of targets). The two mixtures ofspecific vs control targets will be added to each well on a row of 6wells of pre-plated rows of graded number of the CD8(H) or CD8(B) cellswith the same amount of calculated numbers based on the E/T ratio from3:1 to 0.01:1 (well 6-2) while each well 1 on the row is without CD8+ Tcells.

Cell mixtures were cultured for 5-7 days at 37° C., 5% of CO₂ andassessed by FACS analysis in which the CD8(H) and CD8(B) cells weregated out during the analysis. The Ratio of Experimental culture ofspecific target cells and control target cells from well 2-6 of each rowcontaining the graded number of CD8+ T cells as well as the ratio ofControl culture of specific target cells and control target cells fromwell 1 of each row without CD8+ T cells were determined.

Proliferation of the testing cultures on either specific target cells(hHsp60sp loaded B721/E mixed with B721 or TH1 mixed with B721) orcontrol target cells (B7sp loaded B721/E mixed with B721 or TB1 mixedwith B721) in the absence of testing CD8+ T cells are Control cultures(well 1) or in the presence of testing CD8+ T cells are Experimentalcultures, at each of the grading E/T ratios (well 2-6) were quantified.At each E/T ratio, a percentage of specific inhibition (potency) forboth specific or control cultures is calculated based on the quantifiedproliferation of the Experimental ratio and Control ratio as follows:

Specific inhibition (%) of either specific targets or controltargets=(Control ratio−Experimental ratio)/Control ratio×100%.

The control ratio is the ratio of the quantified proliferation of thecontrol target cells (B7sp loaded B721/E mixed with B721 or TB1 mixedwith B721) or the specific target cells (hHsp60sp loaded B721/E mixedwith B721 or TH1 mixed with B721) in the absence of CD8+ T cells (well 1in each row). The Experimental ratio is the quantified proliferationratio of control target cells (B7sp loaded B721/E mixed with B721 or TB1mixed with B721) or the specific target cells (hHsp60sp loaded B721/Emixed with B721 or TH1 mixed with B721) in the presence of the gradedamount of testing CD8+ T cells (at each E/T ration, well 2-6). Aninhibition curve may be generated to show percentages of specificinhibition for the specific target cells or control targets at differentE/T ratios.

The percentage of maximum inhibition (Inhibition Index) for the testingHLA-E restricted CD8+ Treg cells equals to the percentage of the maximuminhibition for the specific target cells subtracted by the percentage ofthe maximum inhibition for the control target cells. The value of theInhibition Index for the testing HLA-E restricted CD8+ Treg cells may beused to determine whether the testing HLA-E restricted CD8+ Treg cellsare defective in a D0 assay and correctable in a D11 assay.

3. Validation

Initial validation of the potency assay was performed and therepresentative data is provided in Table 3 below using the first type oftargets mentioned above. The validation was performed in three healthyindividuals, each had three tests. From our experience, the values amongdifferent individuals vary from person to person in the range of 10-40%.However, for each individual tested, the values are consistent fromdifferent tests as summarized in Table 3.

The % of Max Inhibition (i.e., peak of inhibition curve) of testing CD8+T cells is calculated as: % of Max Inhibition of target cells loadedwith the hHsp60sp peptide (specific target cells; H cells)−% of MaxInhibition in the group of control target cells loaded with the B7peptide (control target cells; B cells).

The E/T ratio of Max Inhibition of testing CD8+ T cells is the ratio oftesting CD8+ T cells to the target cells.

TABLE 3 CD8+ T Cell Inhibition Assay (Potency Assay) Max Max InhibitionInhibition Sample ID Test (%) (E/T ratio) YRK 1 21.2 0.7:1 2 26.6 0.2:13 22.5 0.7:1 JK 1 18.1 0.2:1 2 22.4 0.2:1 3 18.0 0.2:1 PR 1 34.7 0.7:1 233.3 0.7:1 3 35.0 0.2:1

Also see the drawing of the figures of the data for preclinical studieson different indications using the TH1 vs TB1 targets.

F. Release Testing

Release criteria for the intermediate harvested DCs and the finalproduct pDC(H)s have been developed. The harvested DCs were tested formycoplasma and viability. The pDC(H)s were tested for cell count,immunophenotype, mycoplasma, endotoxin, and sterility cultures in finalformulation. Table 4 lists release tests, including release criteria andmethod.

TABLE 4 Release testing specifications and testing methods Release TestCriteria Method Harvested Mycoplasma Testing Negative PCR (Wuxi-Apptec)DC Viability >70% Trypan Blue Exclusion pDC Total DC count >3.0 × 10⁷Manual Endotoxin Testing ≤5EU/kg Endosafe ™ PTS Sterility Testing NoGrowth Bac-T Alert 14 day Immunophenotyping >80% Flow Cytometry TotalCD11C+ (gated on large cells)

Example 3. Stability of pDC(H)s

The final product was stored at ≤−130° C. and used within 12-18 monthsof manufacture. Studies on short-term and long-term product stabilityduring storage and post-thaw are reported in Tables 5-7. Stability wasdetermined by conducting the potency assay (described above) forfunctional determine. Cryopreserved therapeutic agent vials containingpDC(H)s were removed from a LN2 freezer, quickly thawed in a ˜37° C.water bath and placed immediately on ice. The function of the thawedtherapeutic agent (i.e., pDC(H)s) was compared to that of thetherapeutic agent at the time of original harvest by leukapheresis.Three-month stability data for the therapeutic agent are summarized inTable 5. The long-term stability data is summarized in Table 6. Sincethese stability studies were performed on the therapeutic agentgenerated in the research laboratory, we also performed the sameevaluation on the clinical therapeutic agent generated by themanufacturer.

A. Stability after Short-Term Storage

In Table 5, vials of cryopreserved research therapeutic agent werethawed more than three months post-cryopreservation. The potency assaywas performed as described above. The Potency Parameter is the % of netinhibition of the CD8(H) lines versus CD8(B) lines from each subject.Based on our experience, the values among different individuals varyfrom person to person in the range of 10-40%. The % of net inhibition isalso known as inhibition index.

TABLE 5 Short-term stability data Sample Name Thaw Points PotencyParameter SJ1(2T) Harvest 31.1% Subject PR Month 3 31.9% SJ1(2T) Harvest31.7% Subject BC Month 3 28.5% SJ1(3T) Harvest 23.1% Subject IDI Month 326.6% SJ1(3T) Harvest 15.8% Subject LM Month 3 15.7%

B. Stability after Long-Term Storage

In Table 6, vials of cryopreserved research therapeutic agent werethawed more than one year after cryopreservation. The potency assay wasperformed as described above. The Potency Parameter is the % of netinhibition of the CD8(H) lines versus CD8(B) lines from each subject.From our experience, the values among different individuals vary fromperson to person in the range of 10-40%. The results showed that thefunction of the therapeutic agent after 12-18 months freezing remainedsame.

TABLE 6 Long-term stability data Freshly made THE THE THERAPEUTICTHERAPEUTIC AGENT thaw after AGENT freezing Max Date Max Date MaxInhibition Day/ Max Inhibition Day/ Subject Inhibition (E/T Month/Inhibition (E/T Month/ Periods ID (%) ratio) Year (%) ratio) Year(Months) AVR A 21.6 0.2:1 06/04/13 21.3 0.2:1 10/28/14 16 AVR B 25.70.2:1 07/11/13 37.0 0.2:1 10/29/14 15 AVR C 19.6 0.2:1 07/16/13 18.40.2:1 10/23/14 15 YRK 22.6 0.2:1 05/07/12 25.8 0.7:1 10/29/14 29 ID124.6 0.2:1 05/01/12 27.0 0.2:1 10/15/14 29 PR 33.0 0.7:1 05/22/12 41.10.2:1 10/14/14 28 SP 28.9 0.7:1 05/01/12 24.9 0.2:1 10/14/14 29 Sub 20.20.2:1 02/25/13 34.1 0.2:1 10/16/14 20 28-2

C. Stability at Room Temperature

To ensure product stability from the time of thaw to the time ofinfusion, potency assays (described above) were conducted to determinethe amount of time the product remained functional. As summarized inTable 7, the therapeutic agent remains stable after thaw and up to two(2) hours (i.e., 120 minutes) at room temperature.

In Table 7, the percentage (*%) of cells recovered at different timepoints after thaw and up to two (2) hours reflects the stability of thetherapeutic agent product at room temperature. It is calculated as[(Number of pDC(H)s at different time points/Number of cells at 5minutes time point]×100%.

TABLE 7 Stability at room temperature after thaw 5 30 minutes 60 minutes90 minutes 120 minutes (baseline) after thaw after thaw after thaw afterthaw Sample Cell # Cell # Cell # Cell # Cell # ID (×10⁴) %* (×10⁴) %*(×10⁴) %* (×10⁴) %* (×10⁴) %* Sample 1 6.6 100.0 5.1 84.6 5.3 81.5 6.396.9 7.0 107.7 Sample 2 8.0 100.0 10.0 125 8.0 100 8.0 100 9.0 112.5Sample 3 2.1 100.0 2.2 104.8 2.2 104.8 2.2 104.8 2.1 100.0 Sample 4 2.1100.0 1.7 81.0 2.0 95.2 2.1 100.0 1.8 85.7 Sample 5 1.6 100.0 1.4 87.51.4 87.5 1.4 87.5 1.4 87.5 Sample 6 7.0 100.0 8.0 114.3 9.0 128.6 6.085.7 7.0 100.0 Sample 7 5.0 100.0 4.0 80.0 5.0 100.0 5.0 100.0 4.0 80.0

Example 4. Manufacturing Validation Studies

In the process of manufacturing pDC(H)s, various samples were collectedfor QC tests at different stages: initial—leukapheresis product, postFicoll—collected mononuclear cells, post culture—harvested DCs, and thetherapeutic agent—pDC(H)s. For example, the DCs harvested on Day 6 weresubject to QC tests, including Flow Cytometry (CD11c/CD86/CD3),Sterility (BAC-T 14), Mycoplasma and Manual Cell Count. The pDC(H)s weresubject to QC tests, including Flow Cytometry (CD11c/CD3), Sterility(BAC-T 14), Endotoxin (PTS) and Manual Cell Count. A summary of thevalidation test results is shown in Table 8. A summary of the releasingtest results is shown in Table 9. A summary of the potency test resultsfor the therapeutic agent is shown in Table 10.

TABLE 8 Summary of validation test results Validation ValidationValidation Parameter Stage #1 #2 #3 Total TNC processed Initial 1.19 ×10¹⁰ 5.77 × 10⁹ 1 × 10⁹ TNC post Ficoll Post Ficoll 9.81 × 10⁹ 4.51 ×10⁹ 8.81 × 10⁸ % Monocytes Post Ficoll 24.9% 25.0% 21.2% #MonocytesCultured Post Ficoll 3.75 × 10⁸ 4.20 × 10⁸ 1.88 × 10⁸ # Viable DCsrecovered Post Culture 92.6 × 10⁶ 67.5 × 10⁶ 37.0 × 10⁶ % monocyteRecovery Post Culture 24.7% 16.1% 19.6% CDllc (large cell gate) PostCulture 99.6% 99.4% 96.4% CD11C/CD86++ (large cell Post Culture 16.4%30.1% 18.3% gate) CD3 (total gate) Post Culture 30.1% 45.6% 42.6% #Cells Pulsed Post Culture 50 × 10⁶ 50 × 10⁶ 30.0 × 10⁶ % THE THERAPEUTICTHE THERAPEUTIC 80% 100% 100% AGENT recovered AGENT CD11c (large cellgate) THE THERAPEUTIC 99.3% 99.2% 99.3% AGENT CD11c/CD86++ (large cellTHE THERAPEUTIC 14.0% 30.1% 22.2% gate) AGENT CD3 (total gate) THETHERAPEUTIC 17.5% 44.8% 42.6% AGENT

TABLE 9 Summary of release test results Validation Validation ValidationParameter Stage #1 #2 #3 Release Criteria Sterility DC Harvest No GrowthNo No No Growth (14 DAY) Growth Growth Mycoplasma DC Harvest PendingPending Pending Negative (PCR) Sterility THE No No No No Growth (14 DAY)THERAPEUTIC Growth Growth Growth AGENT Endotoxin THE <2EU/kg <2EU/kg<2EU/kg <5EU/kg 50 kg patient THERAPEUTIC AGENT Flow Cytometry THE 99.6%99.4% 99.3% >80% CD11C on large THERAPEUTIC cells AGENT

TABLE 10 Summary of potency test results CD8+ T cell inhibition assay(Potency test) Max Max Max Max Inhibition Inhibition InhibitionInhibition Sample ID (%) (E/T ratio) Sample ID (%) (E/T ratio) *Day 0Assay **Day 11 Assay (freshly isolated CD8+ T (THE THERAPEUTIC AGENTcells as baseline activated CD8+ T information) cells from DFCI-CMCF andValidation #1 Avotres, Inc.) Control (CU) 14.3 0.01:1 Control 19.7 0.2:1(CU) AVR#1(DF) 19.2 0.04:1 AVR#1 15.5 0.2:1 (DF) Validation #2 Control(CU) 28.8 0.2:1 Control (CU) 38.5 0.1:1 AVR#2 (DF) 24.4 0.2:1 AVR#2 (DF)31.7 0.1:1 Validation #3 Control 23.7 0.2:1 Control (CU) 26.1 0.2:1 (CU)AVR#3 (DF) 18.5 0.2:1 AVR#3 (DF) 23.7 0.2:1 *In a DO assay, the % of MaxInhibition is calculated as: % of Max Inhibition in the group of thespecific target cells loaded with the hHsp60sp peptide—% of MaxInhibition in the group of the control target cells loaded with the B7peptide. **In a D11 assay, the % of Max Inhibition is calculated as: %of Max Inhibition in the group of the specific target cells loaded withthe hHsp60sp peptide—% of Max Inhibition in the group of the controltarget cells loaded with the B7 peptide. This value of each group of theCD8+ T cell lines co-cultured with pDC(H)s tested were then normalizedby subtracting the value of the control CD8+ T cell lines co-culturedwith DCs loaded with B7 peptide.

The above validation runs were all performed at large scale. The goal ofthese scale-up experiments was to establish that sufficient therapeuticagent could be manufactured from a starting apheresis collection, usingthe manufacturing process described herein. The results in Table 10demonstrate the reproducibility of the potency assay in validation runs.

Example 5. HLA-E Restricted CD8+ Treg Specificity Assay UsingHLA-E/hHsp60sp Complex Expression Transfectants: TH1 vs TB1

The CD8+ T cell inhibition assay may be used 1) to identify patients whohave a defect in the HLA-E restricted CD8+ T cells and determine if thedefect can be corrected; 2) to evaluate potency or function of thetherapeutic agent; and/or 3) to monitor efficacious duration of thetherapeutic agent after treatment.

The potency of the pDC(H)s as a therapeutic agent prepared as describedin Example 1-E section was measured by assessing specificity ofautologous HLA-E-restricted CD8+ T cell line to the pDC(H)s in a CD8+ Tcell inhibition assay D11 assay).

Materials

The following materials were used in the CD8+ T cell inhibition assay toevaluate the potency of the pDC(H)s:

B7sp peptide (VMAPRTVLL, SEQ ID NO: 2): obtained from GeneScript, Corp.Lyophilized powder. Lot No: 399490030913;

hHsp60sp peptide (QMRPVSRVL, SEQ ID NO: 1): obtained from PolypeptideGroup. Lypohilized powder. Lot No: 1305013R2

PBS: obtained from Cellgro, Cat #46-013-CM;

RPMI 1640 medium: obtained from Cellgro, Cat #15-041-CV;

PBMC: derived from a donor or patient;

GM-CSF: obtained from Miltenyi Biotec, Cat #170-076-112;

IL-4: obtained from Miltenyi Biotec, Cat #170-076-101;

IL-2: obtained from Peprotech, Cat #200-02;

PFA: obtained from Electron Microscopy Sciences, Cat #15741;

Human CD4 Microbeads: obtained from Miltenyi Biotec, Cat #130-045-101;

Human CD8 Microbeads: obtained from Miltenyi Biotec, Cat #130-045-201;

Lymphocyte Separation Medium: obtained from Cellgro, Cat #25-072-CV;

GMP serum-free DC Medium: obtained from Cellgenix, Cat #20801-0500;

DMSO: obtained from Cellgro, Cat # MT 25950CQC;

Human Albumin 25% USP: obtained from Talecris Biotherapeutics, NDC#13533-684-16;

Fetal Bovine Serum: obtained from Atlanta Biologics, Cat # S11150;

GENETICIN® Selective Antibiotic (G418 Sulfate): obtained from Gibco,Cat#10131035;

Anti CD86 biotin: obtained from BD Bio Sciences, Cat #555656;

Anti CD11c PE: obtained from BD Bio Sciences, Cat #555392;

Anti-Streptavidin FITC: obtained from BD Bio Sciences, Cat #554060; and

Six well Falcon tissue culture plate: obtained from Corning, Inc., Cat#353046.

The Target Cell Lines Used (TH1 vs TB1)

In this example, the specific target cells were from a cell line withsurface expression of an HLA-E/Hsp60sp complex (i.e., HLA-E associatedwith the hHsp60sp peptide of SEQ ID NO: 1), and the control target cellswere from a cell line with surface expression of an HLA-E/B7sp complex(i.e., HLA-E associated with the B7sp peptide of SEQ ID NO: 2).

The target cell lines were generated by routine cloning technology, forexample, engineering a fusion construct comprising the HLA-E gene linkeda polynucleotide sequence encoding the hHsp60sp peptide or the B7sppeptide through a flexible linker in a suitable vector, transfecting thevector to a B721 cell line and selecting clones having surfaceexpression of the desired HLA-E/Hsp60sp complex, which transfectant isidentified as “TH1” below, or HLA-E/B7sp complex, which transfectant isidentified as “TB1” below. The fusion construct may also include afluorescent protein such as GFP or the like as an indicator. It isunderstood by a person skilled in the art that any linker known by thoseskill in the art can be used, which may be any short peptide sequence aslong as it does not form a secondary structure that interferes with themain structure of the HLA-E/peptide complex. By way an example, aGly-Ser linker may be used. Typically, the linker between the peptideand the HLA-E may be 10-20 amino acids long.

By way of an example, the peptide-HLA-E fusion construct may beengineered by linking the reading frame of a polynucleotide sequenceencoding the hHsp60sp peptide (or B7sp peptide) and HLA-E gene readingframe through a linker, which can be transfected into a B721 cells togenerate a cell line having surface expression of an HLA-E/Hsp60sp (orHLA-E/B7sp) complex. For example, the specific target cells were cellsexpressing an HLA-E/Hsp60sp complex (TH1, specific target) or HLA-EB7spcomplex (TB1, control target) on the surface of the cells and depositedwith the American Type Culture Collection (ATCC) under the BudapestTreaty having an ATCC Accession No. 127256; and the control target cellswere cells expressing an HLA-E/B7sp complex on the surface of the cellsand deposited with the ATCC under the Budapest Treaty having an ATCCAccession No. 127257.

The prepared specific target cells and the control target cells werethen placed at 26 C.° overnight in the afternoon on −D1 for the D0 assayand D10 for the D11 assay). 0.25 M of the target cells were prepared foreach treatment.

Procedures

1. D0: Process the PBMC from the subject

-   -   1). Isolation of CD8+ T cells from a patient or donor's PBMC to        obtain CD8+ T cells    -   Upon receipt of 60 ml of PBMC from a patient, CD8+ T cells were        purified and tested in a D0 (day 0) assay for baseline        information. The remaining CD8+ T cells were frozen for        generating a CD8+ T cell line with the therapeutic agent after        iDCs were harvested from the DC culture on Day 6 as described in        Example 1.    -   2). Set up a DC culture on D0 which will be harvested to produce        pDC(H)s on D6.    -   Dendritic cells were derived from PBMC cells and were cultured        in 6-well plates with GMP serum-free RPMI, at 37° C., 5% CO₂ for        1-1.5 hours. The wells were gently washed. The non-adherent        cells were washed away and the adherent cells were then cultured        in DC media containing GM-CSF and IL-4 at final concentrations        of 80 ng/mL and 20 ng/mL, respectively.    -   3). Set up the D0 assay

The Day 0 Assay is designed to test the function of the isolated CD8 Tcells without any in vitro activation by pDC(H)s for the purpose todetermine if the testing HLA-E restricted CD8+ Treg cells have a defectin inhibiting proliferation of target cells. Table 11 shows cultures ofCD8+ T cells, including testing CD8+ T cells (XX) and control CD8+ Tcells from a healthy individual (YY), and targets, including TH1 andTB1, at different ratios in plate 1 for the Day 0 (D0) Assay.

(1). Cells prepared:

a. CD8+ T cells, including freshly isolated or thawed from frozen CD8+ Tcells from the testing subject and normal healthy individual as systemcontrol, and b. targets, including TH1 and TB1 which were placed at 26C.° overnight in the afternoon before D0 for the D0 assay and in theafternoon of D10 for the D11 assay). 0.25 M of the target cells wereprepared for each treatment.

(2). Step.1: 0.26 million of freshly isolated CD8+ T cells or 2.6M ofthe PBMC having a 10% CD8+ T cells are obtained. Testing CD8+ T cells,also referred to as testing effector cells, are prepared in 0.8 ml fortwo sets of targets (TH vs TB) and plated at 0.4 ml/well×2 wells as onetest. The testing effector cells are subject to limiting dilution: 0.3mL of the medium plated into Well 1-5 of each row and 0.4 ml of thetesting effector cells were added to each row of last well (well 6) withno additional medium.

0.1 mL of the CD8+ T cell suspension from the last well is added to nextwell to dilute (¼ dilution), except for well 1, which is served ascontrol without the CD8+ T cells.

In the last well, 0.13 million of CD8 cells×75%=0.1 million/well areadded so that the highest E/T ratio were 0.1/0.0375=2.7.

0.26 Million×1 lines=0.26 million/patient in 0.8 ml were needed.

(3). Step 2: 0.25 million of the specific target cells (TH1) and thecontrol target cells (TB1) are mixed with 0.25 million of unloaded B721in 2 ml of complete RPMI 1640 to make a mixture. The mixture is placedinto a 6 wells/row plate at 0.3 ml/well with a target concentration of0.125M/ml×0.3 ml=0.0375 million/well.

TABLE 11 Plate 1 design for D0 assay Targets 0:1 0.01:1 0.04:1 0.17:10.68:1 2.7:1 XX TB1 1 2 3 4 5 6 TH1 7 8 9 10 11 12 YY TB1 13 14 15 16 1718 TH1 19 20 21 22 23 24 Plate 1 is incubated at 37° C., 5% CO₂ for 5-7days (D5-D7).

(4). Analysis by FACS

The effect of the testing CD8+ T cells on the target cells wasdetermined by calculating the ratio between the two types of targetcells. In particular, the ratio between TH/B721 or TB/B721 cells in thepresence or absence of the testing CD8+ T cells was determined as % ofspecific inhibition (potency):

{[the ratio of TH or TB versus B721 cells in control cultures (withoutCD8+ T cells) minus the ratio in experimental cultures (with CD8+ Tcells)]/the ratio in control cultures}×100%.

2. D6: Generation pDC(H) and pDC(B) lines to set up the CD8(H) andCD8(B) lines

-   -   1). Harvest the DCs, loading the peptide/s to generate pDC(H)        and pDC(B) lines:

The DCs harvested on D6 were loaded with the hHsp60sp peptide to makethe therapeutic agent pDC(H)s, or loaded with the B7sp peptide as acontrol, at 50 μM, 37° C. for 2 hours. The peptide loaded DCs were fixedwith 2% of PFA for generating CD8+ T cell lines. The remaining peptideloaded DCs were aliquoted and frozen in liquid nitrogen for future use.

-   -   2) Set up the CD8(H) and CD8(B) cell lines:

The CD8+ T cell lines were generated by co-culturing 1.5-2×10⁶ ofisolated autologous CD8+ cells (thawed from the vials frozen on D0) with0.2-0.3×10⁶ of the therapeutic agent, i.e., autologous DCs loaded withhHsp60sp, pDC(H), or a control, i.e., autologous DCs loaded with B7sp,pDC(B)s, in 1 mL in 48 well plate to set up the lines of CD8(H),activated by the therapeutic agent pDC(H), or CD8(B) (activated by thecontrol agent pDC(B), then add IL-2 in an amount commonly used by thoseskilled in the art, on the second day. The CD8(H) and CD8(B) cells wereculture for another 5 days and harvested on D11 to be tested with knownCD8+ T cells from normal healthy people as a positive control.

3. D11: Harvested the CD8(H) and CD8(B) lines on their D5 cultures andset up the D-11 assay.

-   -   1). Harvested the CD8(H) and CD8(B) lines and plated into a        48-well plate with a graded number for a graded E/T ratio.    -   2). Preparation of target cells: The target essentially        expressing a complex of HLA-E/hHsp60sp (TH1) or HLA-E/B7sp (TB1)        and a GFP protein on their surface by transfecting B721 cells        with genes encoding a fusion protein and a GFP gene. Cell lines        expressing a complex of HLA-E/hHsp60sp (H cells) or HLA-E/B7sp        (B cells) as a control line, can also be prepared in various        forms, for example, targets expressing a complex of        HLA-E/hHsp60sp or HLA-E/B7sp by loading B721/E cell line with an        hHsp60sp or a control peptide B7sp (see example 6 below)    -   3). Set up for Day 11 Assay: The Day 11 Assay was designed to        test if the defect of the CD8+ Treg cells as determined in the        D0 assay could be corrected by the therapeutic agent pDC(H)s.        Table 12 shows cultures of CD8+ T cells triggered with pDC(H)        ex-vivo, or pDC(B) as control, including CD8(H), CD8(Hf),        CD8(Bf) and CD8+ T cells from a healthy individual, and targets,        including TH1 (mixed with B721) and TB1 (Mixed with B721), at        different ratios in plate 2 for the Day 11 Assay.

Plating of the testing CD8 (H) or CD8(B) lines:

The medium was added at 0.3 mL/well into well 1-5 in each row and thetesting CD8+ T cells were already plated as described above.

0.26 million CD8(H) or CD8(B) cells were suspended in 0.8 ml culturemedium. These two effector lines were for one set of targets, TH and TB.TH is specific target cell line. TB is target control cells.

0.4 mL of CD8 was added to each of last well (well 6), and 0.1 mL tonext well to dilute (¼ dilution). In the last well, 0.13 million×75%=0.1million/well, so the highest E/T ratio was 0.1/0.0375=2.7. A total of0.26 million/lines in 0.8 ml for each patient are needed.

The same procedure of adding the targets as for the D0 assay wasrepeated.

TABLE 12 Plate 2 design for D11 assay B721/E Cells loaded with 0:10.01:1 0.04:1 0.17:1 0.68:1 2.7:1 *CD8(H) TB1 1 2 3 4 5 6 TH1 7 8 9 1011 12 **CD8(Hf) TB1 13 14 15 16 17 18 TH1 19 20 21 22 23 24 ***CD8(Bf)TB1 25 26 27 28 29 30 TH1 31 32 33 34 35 36 ****System Control: TestingCD8+ T cells from normal healthy individual TB1 37 38 39 40 41 42 TH1 4344 45 46 47 48 *CD8(H): CD8+ T cell lines generated by co-culturing theCD8+ T cells with hHsp60sp loaded DCs. **CD8(Hf): CD8+ T cell linesgenerated by the CD8+ T cells co-cultured with hHsp60sp loaded and PFAfixed DCs. ***CD8(Bf): CD8+ T cell lines generated by the CD8+ T cellsco-cultured with hB7sp loaded and PFA fixed DC. ****Testing CD8+ T cellsfrom normal healthy individual as in the D0 assay. Plate 2 was incubatedat 37° C., 5% CO₂ for 5-6 days, and then analyze by FACS as describedabove.

Acceptance Criteria

A patient suffering from an autoimmune disease such as type 1 diabetes(T1D) may be treated with the therapeutic agent pDC(H)s according to thepresent invention if the patient has defective CD8+ T cells identifiedby the screening assay (D0 assay) and the defect was corrected by thetherapeutic agent ex vivo (D11 assay) as described herein.

In the D0 assay, the percentage of the maximum inhibition (InhibitionIndex) is calculated as the percentage of the maximum inhibition in thegroup of the target TH minus the percentage of the maximum inhibition inthe group of the target TB (Inhibition Index). A “Inhibition Index”value of <50% of the testing group compared to the Inhibition Indexvalue of the normal healthy control group as Acceptance Criteria foridentifying a defect of the testing CD8+ T cells; whereas in the D11assay, a “Inhibition Index” value of >50% of the testing group comparedto the Inhibition Index value of the normal healthy control group asAcceptance Criteria for correction of the defect of the testing CD8+ Tcells.

Example 6. HLA-E Restricted CD8+ Treg Specificity Assay Using HLA-EExpression Transfectants Loaded with hHsp60sp or B7sp

The potency of the pDC(H)s as a therapeutic agent prepared as describedin Example 1-E section was also measured by assessing specificity ofautologous HLA-E-restricted CD8+ T cell line to the pDC(H)s in a CD8+ Tcell inhibition assay using HLA-E expression transfectants.

The HLA-E expression transfectants (B721/E) were generated for use inthe CD8+ T cell specificity assay in contrast to the HLA-E/Hsp60spcomplex transfectants used in Example 5. HLA-E fusion construct (pDsRed)was engineered by RT-PCR from the human B cell line B721 using thefollowing primers: CCAAGCTTATGGTAGATGGAACCCTCCTTT (SEQ ID: 3) (forward)and GGGGATCCAACAAGCTGTGAGACTCAGACCC (SEQ ID: 4) (reverse). Amplifiedclones in pCR2.1 were fully sequenced. Six independent full-lengthclones representing the HLA-E 101 haplotype but lacking the 3′termination codon were subcloned into the mammalian expression vectorpDsRed-Express-N1 (Clontech Laboratories Inc.), yielding a single openreading frame encoding a fusion protein consisting of HLA-E joined to avariant of the Discosoma species red fluorescent protein (ClontechLaboratories Inc.). The pDsRed-HLA-E construct was introduced into theHLA class I-deficient B cell line B721 by electroporation, and stableclones were selected by subcloning in Geneticin (G418).

The surface expression of HLA-E on B721 cells transfected with HLA-E(B721/E) was tested by exogenously loading the cells with the hHsp60sppeptide, the B7sp peptide, or a control non-HLA-E-binding peptide at 26°C. for 18 hours. The cells were then washed, stained with anti-HLA-E mAb3D-12 followed by Fl-goat anti-mouse Ig, and analyzed on a FACS can flowcytometer and by Cell Quest software (BD). mAb 4D-12 served as control.Any other anti-HLA-E mAb antibodies may serve the purpose describedherein and can be made by a person skilled in the art via routineexperimentation.

To perform the CD8+ T cell specificity assay, freshly isolated CD8+ Tcells were purified from PBMCs, and CD8(H) and CD8(B) lines weregenerated as described in Example 5. HLA-E-transfected cells (B721/E)served as targets were passively loaded with specific peptides—thehHsp60sp peptide and the B7sp peptide, and control non-HLA-E-bindingpeptide—overnight at 26° C. Equal numbers of unlabeled B721/E cellsloaded with a peptide and CFSE labeled parental B721 cells that were notloaded with the peptide were mixed, and testing CD8+ T cells were addedto the targets at graded E/T ratios, from 3:1 to 0.005:1. Thespecificity of the freshly isolated CD8+ T cells was studied bycomparing their inhibition of target B721/E cells loaded with thehHsp60sp peptide versus those loaded with the B7sp peptide or othercontrol peptide/s. In addition, the specificity of the CD8(H) line wascompared with that of the control CD8(B) line. In this regard, theCD8(H) cells established from normal healthy control subject/s testedhad no effect on B721 cells alone or B721 cells pulsed with B7sp peptideor other control peptide/s, indicating that the normal CD8+ T cells hadno effect on these control cell lines. On day 5-6, the cell mixtureswere assessed by FACS analysis, in which the CD8+ T cells were gated outduring the analysis. The ratio between the two types of targets wascalculated and compared in the presence or absence of the CD8+ T cellsto evaluate the effect of testing CD8+ T cells on the targets. Thepercent of specific inhibition was calculated as: {[ratio of loadedB721/E versus unloaded B721 cells in control cultures (without CD8+ Tcells)−ratio in experimental cultures (with CD8+ T cells)]/ratio incontrol cultures}×100. Statistical analysis by 2-tailed Student's t testof the highest percentage of the inhibition (Inhibition Index) was usedto evaluate significant differences among different groups (P<0.05).

Intracellular CEs secreted by the CD8+ T cells was detected. The CD8(H)and CD8(B) lines were generated from healthy individuals as described.The established HLA-E-transfected cells (B721/E) served as targets totrigger the CD8+ T cells and were passively loaded with hHsp60sp peptideovernight at 26° C., and the B7sp peptide served as control. TestingCD8(H) and CD8(B) cells were added to the target B721/E cells loadedwith different peptides at graded E/T ratios, from 3:1 to 0.005:1. Atdifferent time points, 3-color intracellular staining was performed onthe cell mixture with anti-perforin-PE, anti-granzyme A-FITC, andanti-granzyme B-Bio/Cy following the manufacturer's instructions (BD).The cells were assessed by FACS analysis, in which the CD8+ T cells weregated in during the analysis. The CE expression index was calculated asa function of different E/T ratios: ([% of double positive CE-stainedCD8+ T cells from different E/T ratio cultures]−[% of double-positiveCE-stained CD8+ T cells from the CD8+ T cells that were not triggered bythe target cells])/% of double-positive CE-stained CD8+ T cells from theCD8+ T cells that were not triggered by the target cells.

Example 7. Double-Blind, Randomized Study of Safety, Tolerability andEfficacy of the Cell-Based Therapy in Patients with Type 1 Diabetes

This is an ongoing double-blind, randomized, placebo-controlled study toevaluate the safety, tolerability and efficacy to assess the therapeuticagent for type 1 diabetes (T1D). The study includes 25 new-onset T1Dsubjects who have been identified as having: (a) a defect inHLA-E-restricted CD8+ T cell function associated with pancreatic β celldestruction; and (b) evidence that this defect of HLA-E-restricted CD8+T cells can be corrected by in vitro treatment of pDC(H) of theinventive procedure set forth in the present application at, forexample, Example 5.

Nature of the active ingredient (also known as Therapeutic Agent): TheTherapeutic Agent [pDC(H)s] is an individualized preparation of theautologous immature dendritic cells from the patients' adherent primarymonocytes cultured with GM-CSF and IL-4 for 6 days, and loaded passivelywith the hHsp60sp peptide (SEQ ID NO: 1) in vitro and fixed with 2%paraformaldehyde; suspended for intravenous infusion. Preparation of thedosage form for intravenous infusion: The therapeutic agent iscryopreserved in infusible cryomedia in cryopreservation 20 mL bags.Each infusion bag contains between 7×10⁶ and 10×10⁶ cells, 2 mL DMSO, 10mL of 25% HSA and 8 mL of Plasmalyte-A. Three (3) infusion bags wereprepared for each patient. The manufacturing process of the therapeuticagent is described in the present application in great details in, forexample, Example 1.

Route of administration: The infusions are administered intravenously.

Frequency of administration: Three (3) infusions are administered to thepatients approximately at least 21 days apart, preferably 30 (+/−7) daysapart.

Methodology

Each subject is randomized to one of two groups:

-   -   Therapeutic group: 16 subjects to receive the therapeutic agent,        through i.v. administration.    -   Placebo control: 9 subjects to receive placebo infusion solution        (saline and DMSO) only through i.v. administration.

Duration of the study:

There are three pre-defined periods in this study: screening, treatmentand post-treatment follow-up.

Subjects positive for a CD8+ T cell defect correctable in vitro byco-culturing with immature dendritic cells loaded with the hHsp60sppeptide (SEQ ID NO: 1) were identified by HLA-E restricted CD8+ Tregspecificity assay.

-   -   Screening and cell collection period lasted up to 2 months.    -   Treatment period consisted of three doses, each ˜30 days apart        (Baseline, Month 1, Month 2) and a 1-month post-last dose        assessment period (through Month 3). Thus, the treatment period        is defined to be 3 months in duration.    -   Post-treatment follow-up period extended approximately 21        additional months (and thus through month 24 of the study, and        another 24 months follow up).

Patients are treated with three consecutive doses, administered with 30days (+/−7 days) intervals between the doses. The primary time point forassessment and statistical analysis was 3 months post-last dose (Month5), with longer-term follow-up through 22 months post-last dose (month24).

Efficacy (Endpoints):

The objectives focus on assessing treatment safety, the CD8+ T-cellregulatory system and therapeutic outcomes associated with T1D. Thepharmacodynamic effects of the therapeutic agent are assessed over thecourse of the study period.

The endpoints include:

-   -   (a) Assessment of the HLA-E-restricted CD8+ T cell regulatory        activity (“potency assay”)    -   (b) Changes from baseline in the area under the curve (AUC) of        the stimulated C-peptide levels over a 4-hour mixed meal        tolerance test (MMTT)    -   (c) Changes from baseline in HbA1c    -   (d) Change from baseline in insulin usage    -   (e) Changes from baseline in autoantibody levels

The objectives of this study include assessment of changes inpharmacodynamic markers, for example, correcting the function in theHLA-E restricted CD8+ Treg pathway, and improvements of C-peptidelevels, HbA1c and antibody values, as well as Insulin daily usage. Thesample size of 16 subjects treated with the therapeutic agent and 9subjects treated with placebo provides estimates of the mean andstandard deviation of the treatment benefit of the therapeutic agent ascompared to placebo in these endpoints.

Table 13 summarizes the efficacious effects of the therapeutic agent offixed pDC (pDC(H)), using C-peptide level as a readout, measured up to150 days, of all 25 subjects (Treated group x16 vs Placebo group x9)after the administration of the therapeutic agent on D1. The resultsshowed that after 3 months post treatment, the difference between theC-peptide AUC mean value of pDC(H) treated group and that of the placebogroup was statistically significant: P=0.0145, demonstrating that thedosing regimen of the therapeutic preparation was efficacious on thetreated T1D subjects.

Table 14 shows the effectiveness of the treatment on the correction ofthe defect of the dysfunctional HLA-E restricted CD8+ Treg pathway,followed by promising clinical efficacy. Among the first 8 subjectstreated with pDC(H) where we have data beyond d150, nearly 40% of themwere corrected with their dysfunctional HLA-E restricted CD8+ Tregpathway. Among the remaining 60% who were not corrected, 25% of them hadvery low baseline C-peptide and longer time from diagnosis before theywere treated. We thus estimate that if the recipients all startedtreatment earlier after diagnosis with better starting conditions, thepercentage of correction of the defective HLA-E restricted CD8+ Tregpathway may reach >75%.

TABLE 13 The Total C-peptide AUC level in nmol/L (4 hr MMTT) inTreatment (×16) vs. Placebo (×9) at Day 1 and Day 150 after pDC(H)treatment - sorted by % difference at d150 C-peptide Treatment PatientID Age TFD D1 D150 % Diff *pDC (H) T1D-0101-022 39 6.83 0.57 0.9 59.3pDC(H) T1D-0101-002 29 3.55 0.69 1.05 51.6 pDC(H) T1D-0101-031 21 4.210.75 0.85 13.6 pDC(H) T1D-0101-001 20 6.77 0.47 0.49 5.7 pDC(H)T1D-0101-012 37 8.28 1.63 1.68 3.5 pDC(H) T1D-0101-027 17 7.59 0.43 0.43−0.9 pDC(H) T1D-0101-028 17 6.28 0.6 0.58 −2.3 pDC(H) T1D-0101-017 249.89 0.59 0.51 −14.6 pDC(H) T1D-0101-006 28 10.64 0.55 0.44 −20 pDC(H)T1D-0101-011 19 7.98 0.15 0.11 −28 pDC(H) T1D-0101-029 32 10.41 0.040.03 −28.6 pDC(H) T1D-0101-005 20 3.61 0.39 0.26 −33 pDC(H) T1D-0101-00923 11.4 0.22 0.14 −37 pDC(H) T1D-0101-034 48 9.43 0.76 0.45 −41.1 pDC(H)T1D-0101-013 19 7.03 0.49 0.27 −44.2 pDC(H) T1D-0101-023 31 11.99 0.20.11 −45 pDC(H) Mean × 16 D150 26.5 7.9 0.53 0.52 −10.1 PlaceboT1D-0101-004 33 6.31 0.42 0.45 6.8 Placebo T1D-0101-024 24 9.66 0.660.64 −4.1 Placebo T1D-0101-033 24 7.2 0.46 0.4 −13.8 PlaceboT1D-0101-030 35 6.93 0.9 0.71 −20.5 Placebo T1D-0101-016 26 9.23 0.40.31 −23 Placebo T1D-0101-015 33 7 0.65 0.49 −24.4 Placebo T1D-0101-00323 11.01 0.52 0.39 −25.2 Placebo T1D-0101-025 16 11.33 0.85 0.52 −38.5Placebo T1D-0101-008 22 10.74 0.64 0.34 −46.4 Placebo Mean × 9 D150 26.28.8 0.61 0.47 −21 P = 0.0145 *TFD: Time from diagnosis to the first doseof treatment. *pDC(H)-dendritic cell loaded with hHSP60sp and fixed withparaformaldehyde (PFA). Baseline (measured right before the 1^(st)dose), prior to the treatment; D1 - the day of the first dosing. D150(Visit-7) - Five months post the first dosing, primary readout. Theprimary analysis was performed after the last subject completes the D150(Visit-7) study visit. P value based on prespecified Mixed effect Modelfor Repeated Measures (MMRM) A final analysis will be performed afterthe last subject completes the D720 (Visit-11) study visit. The primaryanalysis was performed after the last subject completes the D150(Visit-7) study visit.

TABLE 14 The effectiveness of treatment with pDC(H) on correction ofdefect in the HLA-E restricted CD8+ Treg pathway 1. Among the 8 treatedpatients treated beyond 150 days, the assay showed (+) in subject #2, #6and #12, indicating the dysfunction of the HLA-E restricted CD8+ Tregpathway are well corrected, these three can be identified as goodresponders to the treatment (3/8 = 37.5%). 2. Subject #1, Assay showed(-) from Visit-6 to Visit-11, may be a non-responder (1/8 =12.5%); 3.Subject #5, Assay showed a transient positive sign at Visit-7 to Visit-8and back to (−) from Visit-9 to Visit-11, seemed to be a non-responder(1/8 = 12.5%); 4. Subject #9 (TFD: 11.4 months and low C-peptide: 0.22at starting point) only had some positive sign at Visit-8, behaves likea very low responder (1/8 = 12.5%); 5. Subject #11 (Low C-peptide: 0.15and high daily Insulin 0.75 at starting point), had some positive signat Visti-6 to Visit-8, behaves like a very low responder (1/8 = 12.5%);6. Subject #13 seemed to be a slow responder. Correction started atVisit-7 on D150 time point and lasted to Visit-9 on D360 time point andwe do not have the information on this subject beyond D360 yet. May needlonger time to identify the responsiveness of this subject (1/8 =12.5%). Summary If the recipients were all started early of thetreatment with better starting conditions, the % of the effectiveness oncorrection of the defect of HLA-E restricted CD8+ Treg pathway bytreatment may reach >75%. About the time course of the correction: therelatively precise and general trend and time range need to have morecompleted data to evaluate. D1 (Visit-3)—the day of the first dosing;D150 (Visit-7)—five months post the first dose; D360 (Visit 9)—one yearpost the first dose; D540 (Visit-10)—one year and half post the firstdose; D720 (Visit-11)—two years post the first dose.

In general, the clinical efficacy profiles on the pDC(H) of the first 8treated subjects at their D360-D720 time points basically fall into the“range”, the “scope” and the “pattern” of, and are consistent with, theeffectiveness of the treatment by AVT001 on the correction of the defectof the dysfunctional HLA-E restricted CD8+ Treg pathway (Tables 13-14).Our observations strongly support the notion that correction of thedysfunctional HLA-E restricted CD8+ Treg pathway, as a primary rootcause of T1D, will lead to promising clinical efficacy. Particularly,the clinical efficacy profiles on the pDC(H) treatment of total 25subjects (Tx16 and Px9) at their D150 time point showed impressive andpromising clinical results (Table 13).

Therapy by administration of pDC(H) is to target the common root causeof a variety of autoimmune diseases, including T1D, by stoppingself-destruction by the patients' own immune system for potential cureand prevention of autoimmune diseases. Efficacy will be optimal if thepatients are diagnosed and receive the treatment at a stage where thedamage to their diseased organ/tissues is reversible. However, for thosepatients at later disease stages, this therapy could serve as anecessary therapy to combine with currently available treatments thattarget disease symptoms, or to combine with stem cell transplantationtherapy to replace the diseased organs. In the latter case, even whenthe stem cell transplantation is successfully accomplished, the replacedtissue/organ will be destroyed again by their own immune system if thedefect of HLA-E restricted CD8+ Treg pathway, the common root cause, isnot corrected by the pDC(H) therapy.

Example 8. Treatment of Multiple Sclerosis

It has been found that patients suffering from the autoimmune diseasesnamely multiple sclerosis (MS), including relapsing-remitting multiplesclerosis (RRMS) and secondary-progressive multiple sclerosis (SPMS),and primary-progressive multiple sclerosis (PPMS) have the samecorrectable defect of HLA-E restricted CD8+ Treg cells as that in thepatients suffering from T1D as shown in FIG. 1 , which demonstrates thatthe defect can be corrected ex-vivo by the same therapeutic compositionof the present invention in the same way as used for the T1D patients asdescribed in this application.

Accordingly, the same therapeutic composition may be administered totreat the MS patients with the same dosing regimen as used to treat T1Din Example 7 or a different regiment that is to be adjusted by a personof ordinary skill in the art depending on the patient's physiologicalconditions, age, gender or prognosis of the autoimmune disease.

Example 9. Treatment of Psoriasis

It has been found that patients suffering from the autoimmune diseasesnamely psoriasis (with and without psoriatic arthritis) have the samecorrectable defect of HLA-E restricted CD8+ Treg cells as that in thepatients suffering from T1D as shown in FIG. 2 , which demonstrates thatthe defect can be corrected ex-vivo by the same therapeutic compositionof the present invention in the same way as used to correct the defectof CD8+ Treg cells in the T1D patients, which is described in thisapplication in great details.

Accordingly, the same therapeutic composition may be administered totreat the psoriasis patients with the same dosing regimen as used totreat T1D in Example 7 or a different regimen that is to be adjusted bya person of ordinary skill in the art depending on the patient'sphysiological conditions, age, gender or prognosis of the autoimmunedisease.

Example 10. Treatment of Rheumatoid Arthritis

It has been found that patients suffering from the autoimmune diseasesnamely rheumatoid arthritis (RA) have the same correctable defect ofHLA-E restricted CD8+ Treg cells as that in the patients suffering fromT1D as shown in FIG. 3 , which demonstrates that the defect can becorrected ex-vivo by the same therapeutic composition of the presentinvention in the same way as used for the T1D patients as described inthis application.

Accordingly, the same therapeutic composition may be administered totreat the RA patients with the same dosing regimen as used to treat T1Din Example 7 or a different regimen that is to be adjusted by a personof ordinary skill in the art, depending on the patient's physiologicalconditions, age, gender or prognosis of the autoimmune disease.

Example 11. Treatment of Lupus

It has been found that patients suffering from the autoimmune diseasesnamely lupus including both systemic lupus erythematosus (SLE) andcutaneous lupus erythematosus (CLE) have the same correctable defect ofHLA-E restricted CD8+ Treg cells as that in the patients suffering fromT1D as shown in FIG. 4 , which demonstrates that the defect can becorrected ex-vivo by the same therapeutic composition of the presentinvention in the same way as used for the T1D patients as described inthis application.

Accordingly, the same therapeutic composition may be administered totreat the lupus patients with the same dosing regimen as used to treatT1D in Example 7 or a different regimen that is to be adjusted by aperson of ordinary skill in the art depending on the patient'sphysiological conditions, age, gender or prognosis of the autoimmunedisease.

Example 12. Treatment of Vitiligo

It has been found that patients suffering from the autoimmune diseasesnamely vitiligo including both segmental and non-segmental vitiligo havethe same correctable defect of HLA-E restricted CD8+ Treg cells as thatin the patients suffering from T1D as shown in FIG. 5 , whichdemonstrates that the defect can be corrected ex-vivo by the sametherapeutic composition of the present invention in the same way as usedfor the T1D patients as described in this application.

Accordingly, the same therapeutic composition may be administered totreat the vitiligo patients with the same dosing regimen as used totreat T1D in Example 7 or a different regimen that is to be adjusted bya person of ordinary skill in the art depending on the patient'sphysiological conditions, age, gender or prognosis of the autoimmunedisease.

Example 13. Treatment of Dermatomyositis

It has been found that patients suffering from the autoimmune diseasesnamely dermatomyositis (DM) have the same correctable defect of HLA-Erestricted CD8+ Treg cells as that in the patients suffering from T1D asshown in FIG. 6 , which demonstrates that the defect can be correctedex-vivo by the same therapeutic composition of the present invention inthe same way as used for the T1D patients as described in thisapplication.

Accordingly, the same therapeutic composition may be administered totreat the DM patients with the same dosing regimen as used to treat T1Din Example 7 or a different regimen that is to be adjusted by a personof ordinary skill in the art depending on the patient's physiologicalconditions, age, gender or prognosis of the autoimmune disease.

Example 14. Treatment of Pemphigus

It has been found that patients suffering from the autoimmune diseasesnamely Pemphigus have the same correctable defect of HLA-E restrictedCD8+ Treg cells as that in the patients suffering from T1D as shown inFIG. 7 , which demonstrates that the defect can be corrected ex-vivo bythe same therapeutic composition of the present invention in the sameway as used for the T1D patients as described in this application.

Accordingly, the same therapeutic composition may be administered totreat the Pemphigus patients with the same dosing regimen as used totreat T1D in Example 7 or a different regimen that is to be adjusted bya person of ordinary skill in the art depending on the patient'sphysiological conditions, age, gender or prognosis of the autoimmunedisease.

Example 15. Treatment of Secondary-Progressive Multiple Sclerosis (SPMS)

It has been found that patients suffering from the autoimmune diseasesnamely SPMS have the same correctable defect of HLA-E restricted CD8+Treg cells as that in the patients suffering from T1D as shown in FIG. 8, which demonstrates that the defect can be corrected ex-vivo by thesame therapeutic composition of the present invention in the same way asused for the T1D patients as described in this application.

Accordingly, the same therapeutic composition may be administered totreat the SPMS patients with the same dosing regimen as used to treatT1D in Example 7 or a different regimen that is to be adjusted by aperson of ordinary skill in the art depending on the patient'sphysiological conditions, age, gender or prognosis of the autoimmunedisease.

Example 16. Double-Blind, Randomized Study of Efficacy of the Cell-BasedTherapy in Patients with Early Relapsing Form of Multiple Sclerosis(RMS)/Clinically Isolated Syndrome (CIS)

A 6-month placebo-controlled RMS/CIS Phase 2 study is conducted toinvestigate the efficacy of the therapeutically peptide-loaded dendriticcell (DC) formulation (“Therapeutic Agent”) as described in Example 7 inearly stage of RMS/CIS patients, who are ex vivo responders to thepotency assay. A study (open label) extension phase is also proposed tocompare the pharmacodynamic response. After 6 months, patients in theplacebo group are switched to the Therapeutic Agent in an open labelphase for a further 6 months.

The potency assay, also known as CD8+ T cell inhibition assay, is usedto identify patients having a defect in HLA-E restricted Treg pathway,which defect can be corrected ex vivo, for example, with the TherapeuticAgent as described in Example 1. Only clinical diagnosed MS patientsfulfilling both criteria are considered “ex vivo responders”.

The study includes approximately 90 subjects, who are ex vivo respondersidentified as having a defect in HLA-E-restricted CD8+ T cell functionassociated with neuron cell destruction assessed by clinical detection,which defect can be corrected by, for example, an in vitro procedure setforth in the present application.

Approximately 90 subjects are randomized in a 2:1 ratio for theTherapeutic Agent to Placebo as follows:

-   -   Therapeutic Agent: 60 subjects to receive the Therapeutic Agent        by intravenous infusion administration.    -   Placebo control: 30 subjects to receive placebo infusion        solution only through intravenous infusion administration.

Each subject is subject to a treatment with the Therapeutic Agent orplacebo. Each treatment includes three consecutive doses of theTherapeutic Agent or placebo, which are administered to the subjects byintravenous infusion with one-month (+/−7 days) intervals between twoconsecutive doses, i.e., baseline (Day 1), Month 1, and Month 2.Infusions must be administered at least 21 days apart. The primary timepoint for assessment and statistical analysis of the efficacy of theTherapeutic Agent is 4 months post-last dose (i.e., at Month 6), withlonger-term follow-up through 10 months post-last dose (i.e., at Month12).

Number of subjects (planned):

Up to approximately 90 subjects are randomized (in a 2:1 ratio for theTherapeutic Agent: Placebo), and thus up to approximately 60 subjectsare treated with the Therapeutic Agent and 30 subjects are treated withPlacebo.

Investigational product, dosage and mode of administration:

Nature of the active ingredient: The Therapeutic Agent for each subjectis an individualized preparation of the autologous immature dendriticcells from the subject's adherent primary monocytes, which are culturedwith GM-CSF and IL-4 for 6 days, and loaded passively with a peptidefrom the hHsp60sp (SEQ ID NO: 1) in vitro before being suspended forintravenous infusion into the subject, as described in Examples 2-4.

Formulation of dosage: The Therapeutic Agent is cryopreserved ininfusible cryomedia in cryopreservation 20 mL bags. Each infusion bagcontains between 7×10⁶ and 10×10⁶ cells. Each subject randomized to theTherapeutic Agent has three such bags manufactured for infusion asdescribed above at, for example Examples 2-4. The cryopreserved cellsare manufactured at the Dana Farber Cancer Institute Cell ManipulationCore Facility (DFCI-CMCF), at Harvard, Boston, and are transported tothe site for infusion.

Route of administration: Each infusion is administered intravenouslyover 15-20 minutes.

Frequency of administration: Three (3) infusions are administeredapproximately 30 (+/−7) days apart. Infusions must be at least 21 daysapart.

Duration of treatment: There are three pre-defined periods in thisstudy.

-   -   Screening and cell collection period lasts up to 3 months.    -   Treatment period consists of 3 doses, each about 30 days apart        (e.g., Baseline, Month 1, Month 2). Thus, the treatment period        is defined to be about 2 months in duration. Post-Treatment        Follow-up Period extends approximately 10 additional months (and        thus through Month 12 of the study).

Reference therapy, dosage and mode of administration:

The Placebo consists of 18 mL saline and 2 mL of 10% dimethyl sulfoxide(DMSO) in 25% Human Serum Albumin (HSA) mixture in 20 mL KryoSure bags,while the investigational drug, the Therapeutic Agent, consists of 20 mlof cell suspension in KryoSure bag.

Both the Therapeutic Agent and Placebo are given by similar intravenousinfusion and masked with an opaque sleeve before and duringadministration to remain blinded.

Primary Endpoint (efficacy):

The primary endpoint is new or enlarging T2 lesions at month 6 (withreference to month 3).

There is a total of five scans of new or enlarging T2 lesions at 3, 6,9, 12 months of each subject over the course of the year.

Durability of treatment effect can be assessed by scanning at months 9and 12. The number of new and enlarging T2 lesions on MRI scans ismeasured by the central MRI reading center, which is blinded totreatments.

Secondary Endpoints (efficacy):

New and enlarging T2 new lesions are measured at the end of the studyafter 1 year on MRI compared to new/enlarging T2 at Months 3, 6, 9 and12.

Time to first relapse. Time to first relapse is the period until thefirst relapse is confirmed from the first study cellular therapyinjection.

Assessment of neurofilament light protein (NfL) at Months 3, 6, 9 and12, as compared with its baseline.

Assessment of glial fibrillary acidic protein (GFAP) at Months 3, 6, 9and 12, as compared with its baseline.

Assessment of the HLA-E restricted CD8+ regulatory T cell function(“potency assay”) at Months 3, 6, 9 and 12, as compared with itsbaseline.

Statistical methods:

Sample Size

The sample size is designed to detect a 60% or more reduction in therisk of new/enlarging T2 lesions for each subject calculated as the sumof the new lesions from Months 3 to 6, and to 9 and 12 MRIs with theprimary endpoint at 6 months. The expected ratio of the averagenew/enlarging T2 in the Therapeutic Agent arm is expected to be at most0.40 of that of the Placebo arm. The allocation ratio of subjects to theTherapeutic Agent and Placebo is set at 2 to 1 to provide moreinformation on the results in the Therapeutic Agent arm as well asproviding a greater incentive for patients to enroll. Subjects in thePlacebo arm are allowed to cross-over to the Therapeutic Agent at 6months. The Alpha (the probability of rejecting the null hypothesis whenit is true) is 0.05. The Power is set at 80% (i.e., the probability ofrejecting the null hypothesis when it is false). The sample size iscalculated based on the ratio of two negative binomial new/enlarging T2rates using the Wald Test (PASS Version 14.0.14).

The null and alternative Hypotheses are:

H₀: RR=1 vs. H_(a): RR≠1

The Posenimode Phase 2 study showed an average of 0.7 new/enlarging T2total over three MRIs or 0.23 per scan; the average per lesion countfrom the Phase 2 Oftatumamab trial of 1.04 new/enlarging T2 over 3 scansor 0.35 per scan and the Ozanimod Phase 2 trial showed an average of 8.6over 5 scans or 1.7 per scan. Given that this is a Placebo controlledtrial, it is possible that subjects with the most severe MRIs in termsof lesion counts may be excluded. Thus, we reduce the expected averagecount to be conservative. We assume that the Placebo arm experiences anaverage of 3 new/enlarging T2 lesions at 6 months. If the number ofnew/enlarging T2 lesions is higher, the power is increased. Toaccommodate the skewness often seen in lesion counts, a negativebinomial distribution is used with a dispersion parameter set to 1.2.For a two-sided Wald test of the null hypothesis H0: RR=1 vs. thealternative Ha: RR≠1, samples of 25 subjects in the Placebo Group and 50subjects in the Therapeutic Agent Group achieve 81% power to detect anevent rate ratio (RR) of 0.40 or smaller, when the event rate in thePlacebo Group (A1) is 3 new/enlarging T2, the average exposure time inboth groups (p(t)) is 6 months and the two-sided significance level(alpha) is 0.05 with the null hypothesis variance calculated usingmaximum likelihood estimation. Although we expect few dropouts given the6-month duration of the study, we increase the sample size toaccommodate up to 15% dropouts. Thus 75/0.85=88.2 or 90 subjects arerandomized with 30 assigned to the Placebo Group and 60 assigned to theTherapeutic Agent Group.

Analysis Timing

The primary analysis for this study is performed after the last subjecthas completed the Month 6 study visit, at which time the study databaseis locked, and the treatment allocation codes unblinded for analysis.These analyses form the primary basis for the assessment of the studyobjectives.

Continuing data collection through the long-term follow-up is performedthrough Month 12 of the study.

Efficacy Assessment:

New and/or enlarging T2 lesions measured by MRI (new/enlarging T2),after 6 months are assessed along with the secondary objectives.

Primary Analyses:

The primary analysis compares the ratio of risk of new/enlarging T2using the negative binomial distribution via PROC GENMOD in SAS (version9.4 or higher) with covariates of age, sex, baseline lesion count(Gadolinium plus T2 lesions) and treatment group. The analysis utilizesthe new/enlarging T2 over the period 3 to 6 months.

Secondary Analyses:

Durability of treatment is assessed by examining the change from thenew/enlarging T2 at 6 months in the Therapeutic Agent randomized groupto the new/enlarging T2 at the month 12 MRI. The durability is assessedby whether or not the change is within a 90% confidence interval of theestimated mean count of new/enlarging T2 at month 6 from within theTherapeutic Agent Group.

The negative binomial distribution is also used to assess the annualizedrelapse rate as done for the primary. Baseline lesion counts are used asa covariate instead of prior relapses since these are naïve subjectsunlikely to have full relapse histories. Time to first relapse isassessed using a Cox model with the same covariates and a descriptiveanalysis using a Kaplan Meier analysis. The laboratory values NfL, GFAP,and CD8+ cells are assessed using the log of the values using mixedmodels repeated measures.

All documents, books, manuals, papers, patents, published patentapplications, guides, abstracts, and/or other references cited hereinare incorporated by reference in their entirety. Other embodiments ofthe invention will be apparent to those skilled in the art fromconsideration of the specification and practice of the inventiondisclosed herein. It is intended that the specification and examples beconsidered as exemplary only, with the true scope and spirit of theinvention being indicated by the following claims.

What is claimed:
 1. A composition comprising dendritic cells loaded withhHsp60sp, wherein the dendritic cells are from a subject in need of thedendritic cells loaded with hHsp60sp, and the dendritic cells loadedwith hHsp60sp have been fixed with paraformaldehyde (PFA).
 2. Thecomposition of claim 1, wherein the dendritic cells loaded with hHsp60spare in a therapeutically effective amount for correcting correctabledefect of HLA-E restricted CD8+ Treg cells from a subject.
 3. Thecomposition of claim 1, wherein the dendritic cells loaded with hHsp60spare in a therapeutically effective amount for treating an autoimmunedisease in a subject.
 4. The composition of claim 1, further comprisinga medium.
 5. The composition of claim 4, wherein the medium comprisesdimethyl sulfoxide (DMSO), human serum albumin (HAS) and plasmalyte-A.6. The composition of claim 1, further comprising greater than 80% totalCD11c+(gated on large cells).
 7. The composition of claim 1, furthercomprising a cryoprotectant.
 8. The composition of claim 1, wherein thecomposition is formulated for intravenous administration to the subject.9. The composition of claim 1, wherein the subject suffers from anautoimmune disease.
 10. The composition of claim 9, wherein theautoimmune disease is selected from the group consisting of type 1diabetes (T1D), multiple sclerosis (MS), psoriasis, rheumatoidarthritis, lupus, vitiligo, pemphigus and dermatomyositis.
 11. Thecomposition of claim 9, wherein the autoimmune disease is type 1diabetes (T1D).
 12. The composition of claim 9, wherein the autoimmunedisease is multiple sclerosis (MS).
 13. A method for preparing acomposition, the method comprising: (a) isolating mononuclear cells froma subject; (b) incubating the mononuclear cells in a culture for no morethan six days to produce immature dendritic cells (DCs); (c) harvestingthe immature DCs from the culture in step (b); (d) incubating theharvested DCs with hHsp60sp to produce hHsp60sp loaded dendritic cells(DCs); (e) fixing the hHsp60sp loaded DCs with paraformaldehyde (PFA) toproduce fixed hHsp60sp loaded DCs; and (f) suspending the hHsp60sploaded DCs in a medium, whereby the composition is prepared.
 14. Themethod of claim 13, wherein the hHsp60sp loaded DCs are fixed with 2%PFA in step (d).
 15. The method of claim 13, wherein the composition isformulated for intravenous administration to the subject.
 16. The methodof claim 15, wherein the subject suffers from an autoimmune disease. 17.The method of claim 16, wherein the autoimmune disease is selected fromthe group consisting of type 1 diabetes (T1D), multiple sclerosis (MS),psoriasis, rheumatoid arthritis, lupus, vitiligo, pemphigus anddermatomyositis.
 18. The method of claim 16, wherein the autoimmunedisease is type 1 diabetes (T1D).
 19. The method of claim 16, whereinthe autoimmune disease is multiple sclerosis (MS).
 20. A compositionprepared according to the method of claim 13.